Method for detecting radiation exposure

ABSTRACT

A method is disclosed for detecting exposure of organisms to biologically significant or hazardous amounts of ionizing radiation. The method uses nucleic acid microarray hybridization to evaluate biological effects, such as patterns of expression of genes after radiation exposure. Numerous genes are provided which have been found to be responsive to radiation exposure in a variety of cell lines. These genes are incorporated into probe sets, which are exposed to a labeled nucleic acid composition from a test cell, such as cDNA reverse transcribed from mRNA in the test cell, which specifically hybridizes to members of the probe set when the cell has been exposed to a biologically significant amount of ionizing radiation. Whether the nucleic acid composition hybridizes to the nucleic acid molecules representing genes that are differentially expressed is determined. The invention also includes methods for determining a dose response relationship between radiation exposure and differential expression of one or more genes, for example to determine a probable radiation dose in cells that have actually or potentially been exposed to the ionizing radiation. The invention also includes probe sets and microarrays used in this method.

This is a divisional of U.S. application Ser. No. 09/913,171, filed Aug.9, 2001, now U.S. Pat. No. 7,008,768 issued Mar. 7, 2006, which is aU.S. national stage § 371 application of PCT/US00/04897 filed Feb. 25,2000, which was published in English under PCT Article 21(2), whichclaims the benefit of U.S. provisional application No. 60/121,756 filedFeb. 26, 1999.

FIELD

The present invention concerns gene expression, and is related to thedetection of differential gene expression following exposure of cells toionizing radiation.

BACKGROUND

Ionizing radiation has many medical, industrial and military uses.Although ionizing radiation can be used in the therapy of diseases suchas cancer, exposure to biologically significant levels of such radiationcan also cause genotoxic stress. Similarly, many industrial processes(such as the production of nuclear power) and military uses (such asnuclear weapons) can expose individuals to hazardous levels of ionizingradiation. Such radiation can elicit a variety of cellular responses,ranging from cell-cycle arrest to mutation, malignant transformation, orcell death. Many of the responses (such as genotoxicity) are oftensubtle, and exposed persons may be unaware or unsure if they have beenexposed. Moreover, it may require years to evince an untoward effect(such as the development of a malignancy) caused by the exposure.

Many of the assumptions about low dose effects have been based onextrapolations from effects measured at high doses. Transcriptionalresponses to doses of ionizing radiation with relatively little effecton cell survival have not been as well investigated, although smallvariations in expression levels of several isolated genes have beendetected. A dose of 50 cGy reportedly reduced expression of β- andγ-actin (Woloschakand Chang-Liu, Int. J. Radiat. Biol. 59:1173-83, 1991)and induced RB-1 and H4 histone (Woloschak and Chang-Liu, Cancer Lett.97:169-75, 1995) in Syrian Hamster Embryo cells, while a decrease inc-myc and increase in c-jun was detected in these cells following a doseas low as 6 cGy (Woloschak and Chang-Liu, Cancer Lett. 97:169-75, 1995).In a transformed human lymphoblast cell line, activation of NF-κB hasbeen reported with as little as 10 cGy of radiation (Prasad et al.,Radiat. Res. 138:367-72, 1994), along with induction of c-FOS, c-JUN,c-MYC and c-Ha-RAS in the 25-200 cGy range (Prasad et al., Radiat. Res.143:263-72, 1995). The induction by 25 cGy of PBP74, a member of theheat shock 70 gene family, has also been reported in two human cancercell lines (Sadekova et al., Int. J. Radiat. Biol. 72:653-60, 1997).

SUMMARY

It would be advantageous to have a method for detecting exposure oforganisms to biologically significant or hazardous amounts of ionizingradiation. Although small variations in expression levels of severalisolated genes in cell lines have been detected at lower doses, none ofthese studies have demonstrated a dose-response relationship for geneinduction at low radiation doses, and overall qualitative and/orquantitative patterns of differential expression have not beeninvestigated. The present invention uses nucleic acid microarrayhybridization to evaluate biological effects, such as patterns ofexpression of genes after radiation exposure. Using these methods,numerous genes have been found which are responsive to radiationexposure in a variety of cell lines, and microarrays have beenconstructed which are capable of detecting biological responses (such aspatterns of expression) to radiation exposure with great sensitivity andspecificity.

The present invention includes a method of identifying cells that havebeen exposed to radiation induced biological stress. The method furtherincludes providing a probe set that includes nucleic acid moleculesrepresenting genes that are differentially expressed in cells that havebeen exposed to a biologically significant amount of ionizing radiation.The probe set is exposed to a labeled nucleic acid composition from atest cell which specifically hybridizes to members of the probe set whenthe cell has been exposed to a biologically significant amount ofionizing radiation. Whether the nucleic acid composition hybridizes tothe nucleic acid molecules representing genes that are differentiallyexpressed is determined.

The probe set may be nucleic acid molecules (such as cDNAs oroligonucleotides) bound in an array to a surface, wherein thenucleotides specifically hybridize to sequences in the nucleic acidcomposition from the test cell. In one example, the nucleic acidcomposition includes cDNA reverse transcribed from mRNA in the testcell, and labeled with a fluorophore that detects hybridization of thecDNA to the probe set. In another example, the method also includesexposing the probe set to a labeled nucleic acid composition from acontrol cell which has not been exposed to a biologically significantamount of ionizing radiation. Genes which are expressed in the absenceof radiation exposure will therefore produce mRNA from which labeledcDNA is made that specifically hybridizes to some members of the probeset. The nucleic acids from the test cell and control cell can belabeled with different signals (such as red and green colors) toindicate differential (either increased or decreased) expression ofgenes in the test cell as compared to the control cell.

The probe set may include probes that specifically hybridize to thelabeled nucleic acid composition from specimens obtained more than fourhours after exposure to the biologically significant amount of ionizingradiation, and/or less than 24 hours after exposure. In anotherembodiment of the invention, the probe set includes probes thatspecifically hybridize to the labeled nucleic acid composition fromspecimens obtained more than 24 or 48 hours after exposure to thebiologically significant amount of ionizing radiation. Probes whichdetect such late effect exposures may be used to screen for radiationexposure when such screening is not done until one or two days followingpotential radiation exposure, when a subject is examined in a medical orlaboratory facility.

In yet other embodiments of the invention, the probe set includes probesthat specifically hybridize to the labeled nucleic acid composition fromspecimens which have been exposed to less than about 25 cGy of ionizingradiation. The probe set may also include genes that are differentiallyexpressed by at least 1.5-fold or 2-fold following exposure to abiologically significant amount of ionizing radiation. The probe set mayinclude at least 10%, 30% 40%, 50%, 75%, 80%, 90%, 95%, or 99% of theprobes identified in Tables 9, 10, 11, 12, 13 or 14 or the entire probeset shown in any of those Tables. The probe set may also include atleast 10 or 20 of the probes identified in Tables 9, 10, 11, 12, 13 or14. Examples of probes that represent such late effects include thoselisted in Tables 9-12. In another embodiment, the probe set includesnucleic acid sequences that are selected for having differentialexpression following exposure to a biologically significant amount ofionizing radiation. The probe set may be at least 50%, 75%, 80%, 90%,95%, 99%, or consist essentially of nucleic acid sequences that aredifferentially expressed following exposure to a biologicallysignificant amount of ionizing radiation. In yet another embodiment, theprobe set includes nucleic acid sequences that are selected for having adifferential expression of at least 1.5- or 2-fold following exposure toa biologically significant amount of ionizing radiation.

In yet other embodiments of the method, a plurality of nucleic acidprobe elements are bound to a surface, for example in an array, whereinthe nucleic acid represents a gene product (including a protein or anucleic acid such as RNA) that is differentially expressed by a cellfollowing radiation induced biological stress. The plurality of probeelements are contacted with a plurality of gene products from a testcell, under conditions that allow the gene products (such as the nucleicacid sequences) to specifically hybridize to one of more of the probeelements, and provide a signal which indicates differential expressionof one or more genes in a test cell has been exposed to biologicallysignificant levels of ionizing radiation, and detecting the presence orabsence of the signal. The probe elements may be selected from a set ofnucleic acids that specifically hybridize to nucleic acids obtained fromcells exposed to ionizing radiation. For example, the target elementsare nucleic acid sequences that are differentially expressed by a cellmore than 4, 24 or even 48 hours after exposure to the ionizingradiation. The probe elements may also include, or be limited to,nucleic acid sequences that are differentially expressed by at least1.5-fold or 2-fold following exposure to a biologically significantamount of ionizing radiation.

The target elements may be one or more of the clones listed in Tables 9,10, 11, 12, 13, or 14, for example Image ID clones 39993, 47475, 109123,120362, 136114, 195365, 202549, 209340, 221846, 232837, 241412, 244227,251516, 260619, 280386, 297442, 308588, 549146, 753418, 841278, 51699,417226 and 28116. The probe nucleic acids may be DNA, such as cDNA, andcDNA obtained from mRNA expressed by the test cell. When the probe isreverse transcribed from cellular RNA, it may average about 1000-2000nucleotides in length, but may in some instances be as long as 10,000nucleotides. The probe nucleic acids of the probe set may be about asshort as 8 or 10 nucleotides in length, but may also be as long as about1000 to 1,000,000 nucleotides in length.

The method can also include contacting the probe elements with aplurality of control nucleic acids obtained from mRNA (for example byreverse transcription) of a control cell that has not been exposed tobiologically significant levels of ionizing radiation and determiningwhether the nucleic acids from mRNA of the control cells hybridizedifferentially to the probe elements than the nucleic acid compositionfrom the test cell. The test nucleic acid sequences are labeled with afirst label that detects hybridization of the test nucleic acidsequences to the probe sequences, and the control nucleic acid sequencesare labeled with a second label that detects hybridization of thecontrol nucleic acid sequences to the probe sequences. The first andsecond labels interact to indicate whether expression of a nucleic acidsequence in the test cell has increased or decreased, relative to abaseline level. The first and second labels may be fluorophores ofdifferent colors. The nucleic acids from the control cells may, forexample, be labeled with a green fluorophore, and the nucleic acids fromthe test cells may be labeled with a red fluorophore. Hence targetelements for which differential gene expression does not occur willappear yellow, while underexpressed (decreased) gene expression will beindicated by green and overexpression (increased expression) by red.

The test cells may be animal cells, such as human cells, for examplehuman peripheral blood cells, for example peripheral blood mononuclearcells, such as lymphocytes. In addition, the cells may be microbial orplant cells, such as microbes or cells from plants in the vicinity of asuspected environmental exposure to ionizing radiation.

In view of the set of stress response genes which have been identified,and may be identified using the present methods, the invention alsoincludes methods of making microarrays for identifying cells that haveactually or potentially been exposed to a biologically significantamount of ionizing radiation, by identifying genes that aredifferentially expressed by a cell following exposure to biologicallysignificant amounts of ionizing radiation. A probe set is then provided,each element of the set including a nucleic acid sequence from a genethat is identified as differentially expressed by a cell followingradiation induced biological stress. The target nucleic acid sequence iscapable of hybridizing to a nucleic acid sequence which isdifferentially expressed by the cell following exposure to thebiologically significant amount of ionizing radiation. In otherembodiments, the genes that are differentially expressed by a cell areidentified by exposing the cell to a biologically significant amount ofionizing radiation, obtaining mRNA expressed by the cell, reversetranscribing the mRNA into cDNA, labeling the cDNA, and hybridizing thelabeled cDNA to a probe set that represents potential genes that may bedifferentially expressed and identifying members of the probe set thathybridize with the labeled cDNA. Any high throughput genomic analysismay be used to analyze the differential expression of the stressresponse genes, as may more standard molecular biology techniques suchas dot-blot hybridization. The genes may include p53 regulated genes.

In another embodiment, the method further includes determining a doseresponse relationship between radiation exposure and differentialexpression of one or more genes, for example to determine a probableradiation dose in cells that have actually or potentially been exposedto the ionizing radiation. In yet another embodiment, identifying genesthat are differentially expressed, for making a microarray, includesidentifying genes that are differentially expressed in a cell type thatis to be obtained from a subject for testing. The microarray may be usedto measure a biological response to potential radiation exposure in thesubject, for example in a cell type. The invention also includesmicroarrays which are made by this method.

The cell type may be peripheral blood cells, for example peripheralblood mononuclear cells, such as lymphocytes. In addition, the cell typemay be any microbial or plant cell.

The invention also includes a method of diagnosing biologicallysignificant radiation exposure in a subject, by obtaining a biologicalspecimen from the subject, synthesizing cDNA from mRNA expressed in oneor more cells of the biological specimen, and labeling the mRNA with adetectable label. The labeled mRNA is exposed to a probe set whichrepresents genes that are differentially expressed in the biologicalspecimen following exposure to the radiation. A determination is thenmade whether the labeled mRNA selectively hybridizes to one or moreprobes of the probe set that are associated with the radiation exposure,or hybridizes in a pattern that is associated with radiation exposure.Particular patterns of hybridization can also be associated withspecified exposure doses, or time periods following exposure. The probeset may be one or more of the probes listed in any of Tables 9, 10, 11,12, 13 or 14, or a probe set that includes at least 10%, 30% 40%, 50%,75%, 80%, 90%, 95%, or 99% of the probes listed in any of Tables 9, 10,11, 12, 13 or 14. In another embodiment, the method detects patterns ofdifferential expression associated with biologically significantradiation exposure.

The invention also includes use of the microarrays of the presentinvention for measuring a biological response to in a subject, byobtaining a biological sample from the subject, synthesizing cDNA frommRNA expressed in one or more cells of the biological sample, labelingthe mRNA with a detectable label, and exposing the labeled mRNA to aprobe set which represents genes that are differentially expressed inthe biological sample following radiation exposure, and determining ifthe labeled mRNA selectively hybridizes to one or more probes of theprobe set that are associated with radiation exposure. The subject maybe undergoing radiotherapy (or a candidate for radiotherapy) for thetreatment of cancer, and the microarray can used to monitor or predictthe subject's biological response to the radiotherapy.

Also included in the invention are the probe sets that provideinformation about exposure to biologically significant doses of ionizingradiation, for example probe sets including the DNA probe sets shown inany of Tables 9, 10, 11, 12, 13 or 14, or subsets of the probe sets ofTables 13 or Table 14. Such subsets may include sets having at least10%, 30% 40%, 50%, 75%, 80%, 90%, 95%, or 99% of the probes sets shownin any of Tables 9, 10, 11, 12, 13 or 14.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a graph showing the clonal survival of ML-1 cells exposed to¹³⁷CS γ-rays. Points are the average of five independent experiments,and error bars are standard errors. Linear regression of the data wasused to fit the curve shown.

FIG. 2 is a bar graph showing the percent of apoptotic cells one or twodays after radiation treatment. Results are the average of 3-4independent experiments in which at least 250 cells were scored for eachpoint. Error bars are standard errors.

FIG. 3A is a graph showing the fraction of S-phase cells in thepopulation at various times after irradiation of exponentially growingML-1 cells with 2 cGy (▪), 5 cGy (⋄), 10 cGy (●), 25 cGy (Δ), or 250 cGy(▾) of ¹³⁷Cs γ-rays. Points are the mean of three independentexperiments, and error bars are standard deviations. The points for the12 and 24 hour timepoints have been offset slightly to make the errorbars more distinguishable. The shaded area represents the range ofS-phase fractions in untreated control populations and is the mean plusand minus the standard deviation of the control measurements at alltimepoints.

FIG. 3 B is a bar graph showing the cell cycle distribution at the timeof maximum perturbation (12 hours) for the low dose treatments. For eachdose, the percent of cells in G1 is represented by the open bar, cellsin S by the grey bar, and cells in G2 by the black bar. The values shownare the mean of three independent experiments, and error bars arestandard deviations. Where error bars are not visible the standarddeviation was ≦0.76. An asterisk indicates a point not significantlydifferent from the control at or below the p<0.005 level.

FIG. 4 is a schematic drawing of a quadrant of a microarray hybridizedto RNA from untreated ML-1 cells (labeled with a green fluorophore,represented as circles containing horizontal lines) and ML-1 cells fourhours after treatment with 20 Gy¹³⁷ (labeled with a red fluorophore,represented as circles containing vertical lines). Targets appearing asyellow spots (represented as circles containing horizontal and verticallines) have equal representation of both fluorophores and indicate nochange in expression by the irradiation. Red spots (circles containingvertical lines) are targets associated with increased expressionfollowing irradiation, and green spots (circles containing horizontallines) are associated with decreased expression. Blank circles representareas where no hybridization occurred. A horizontal row of green dots(circles containing horizontal lines) at the top and bottom of theschematic microarray represent DNA labeled prior to printing on thearray, and serve as orientation markers for a computerized scanner.

FIGS. 5A and 5B are schematic diagrams illustrating increased ordecreased expression of transcripts identified on a microarray. Theexpression was measured in a panel of human cancer cell lines listedalong the left margin, where the cell lines are shown divided into p53wild type cells and p53 mutant cells. Numbers shown in the diagrammeasure relative induction for each gene over levels in untreatedcontrols four hours after treatment with a 20 Gy dose of (A) ionizingradiation or (B) following treatment with 100 μg/ml methylmethanesulfonate (MMS) or 14 J/m² ultraviolet (UV) radiation. A zeroindicates no detectable expression in either control or treated cells.The results are coded: red (vertical lines) for >2-fold induction, green(horizontal lines) for >2-fold reduction, and yellow (horizontal andvertical lines) for <2-fold change from untreated control.

FIGS. 6A and 6B are graphical representations showing the time-course ofinduction of the (A) CIP1/WAF1 and (B) GADD45 genes followingirradiation by 2 cGy (▪), 5 cGy (⋄), 10 cGy (●), 25 cGy (Δ), or 50 cGy(▾) of γ-rays. Data is shown from a representative experiment. Thedashed line indicates the basal level in untreated controls.

FIGS. 7A and 7B are graphical representations showing the maximalinduction of (A) CIP1/WAF1 (◯), GADD45 (▪) and (B) MDM2 (▪), ATF3 (◯),and BAX (♦) by low doses of γ-rays. Points are the average of 4independent experiments, and error bars are standard errors. The dashedline indicates the basal level in untreated controls, solid lines werefit by linear regression through the data.

FIG. 8A is a graphical representation showing the dose response ofmaximal induction of CIP1/WAF1 across a broader range of doses. Data isshown from a representative experiment.

FIG. 8B is a bar graph showing the relative induction of CIP1/WAF1 andGADD45 which would be required per lethal event if the gene inductionmeasured at low doses occurred only in lethally damaged cells. If onlylethally damaged cells contributed to the observed gene induction, theinduction per lethal event would be expected to be a constant for alldoses. As this is not the case, it suggests surviving cells docontribute significantly to gene induction.

FIGS. 9A and 9B are bar graphs showing the induction of (A) ATF3 in p53wild-type RKO cells (light bar), RKO/E6 cells (dark bar) and the thymusof p53 wild-type mouse (light bar), or p53−/− (dark bar); and (B) FRA-1in p53 wild-type MCF7 cells (light bar), or in MCF7/E6 cells (dark bar)and in the thymus of p53 wild-type mouse (light bar), or p53−/− mouse(dark bar). The results shown for the RKO cell lines are the average of4 independent experiments, and error bars are standard errors of themeans. The results for the MCF7 cell lines are the average of 3independent experiments.

FIG. 10 is a bar graphic representation of a section of a microarrayhybridized to RNA from unirradiated (filled bars, normalized to thelevels in the first donor) and 2 Gy gamma-irradiated (open bars) humanperipheral blood lymphocytes harvested 24 hours after irradiation.Results are from 4-8 independent donors. Error bars are standarddeviations.

FIG. 11 is a graphical representation showing a timecourse of markerlevels following 2 Gy gamma-rays delivered ex vivo. Each point is themean of relative induction above background in 3-4 independent donorsfor DDB2 (▪), CIP1/WAF1 (♦), XP-C (●), PCNA(□) and IFNγp10 (◯). RNA washarvested from lymphocytes following one, two, or three days ofincubation. Error bars are standard errors of the mean. The dashed lineshows the basal level of expression in untreated PBLs.

FIGS. 12A-D are graphical representations showing the dose-responserelationship for the induction of DDB2 (▪), CIP1/WAF1 (♦), and XP-C (●)in human PBL. PBL were irradiated ex vivo with graded doses of γ-raysand RNA was harvested after (A) 4 hours, (B) 24 hours or (C) 48 hours(D) 72 hours of incubation. Points are the mean of relative expressionin three or more independent donors, and error bars are standard errorsof the mean. The dashed lines indicate the level of expression inun-irradiated control cultures.

SEQUENCE LISTING

SEQ ID NO: 1 is the nucleic acid sequence of Image ID Number 26474.

SEQ ID NO: 2 is the nucleic acid sequence of Image ID Number 268652.

SEQ ID NO: 3 is the nucleic acid sequence of Image ID Number 34396.

SEQ ID NO: 4 is the nucleic acid sequence of Image ID Number 35318.

SEQ ID NO: 5 is the nucleic acid sequence of Image ID Number 50615.

SEQ ID NO: 6 is the nucleic acid sequence of Image ID Number 110589.

SEQ ID NO: 7 is the nucleic acid sequence of Image ID Number 203132.

SEQ ID NO: 8 is the nucleic acid sequence of Image ID Number 247483.

SEQ ID NO: 9 is the nucleic acid sequence of Image ID Number 251591.

SEQ ID NO: 10 is the nucleic acid sequence of Image ID Number 360778.

SEQ ID NO: 11 is the nucleic acid sequence of Image ID Number 415817.

SEQ ID NO: 12 is the nucleic acid sequence of Image ID Number 469954.

SEQ ID NO: 13 is the nucleic acid sequence of Image ID Number 489805.

SEQ ID NO: 14 is the nucleic acid sequence of Image ID Number 753447.

SEQ ID NO: 15 is the nucleic acid sequence of Image ID Number 884719.

SEQ ID NO: 16 is the nucleic acid sequence of Image ID Number 1493160.

DETAILED DESCRIPTION OF SEVERAL EMBODIMENTS Abbreviations andDefinitions

The following definitions and methods are provided to better define thepresent invention and to guide those of ordinary skill in the art in thepractice of the present invention. It must be noted that as used hereinand in the appended claims, the singular forms “a” or “an” or “the”include plural referents unless the context clearly dictates otherwise.Thus, for example, reference to “a protein” includes a plurality of suchproteins and reference to “the array” includes reference to one or morearrays and equivalents thereof known to those skilled in the art, and soforth.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood to one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. The materials, methods, and examples areillustrative only and are not intended to be limiting.

MMS: methanesulfonate

RT: room temperature

Animal Cells: Cells obtained from multicellular vertebrate organisms, acategory which includes, for example: mammals, primates, rodents,veterinary subjects, and birds. The cells can be obtained from anysource, for example peripheral blood, urine, saliva, tissue biopsy,surgical specimen, amniocentesis samples and autopsy material. Fromthese cells, genomic DNA, cDNA, mRNA, RNA, or protein can be isolated.

Biologically significant radiation exposure: An amount of radiationexposure sufficient to cause differential expression of stress genes incells after they are exposed to the radiation.

Biological Specimen/Sample: One or more cells obtained from an animal orplant.

Cancer: malignant neoplasm that has undergone characteristic anaplasiawith loss of differentiation, increased rate of growth, invasion ofsurrounding tissue, and is capable of metastasis.

cDNA (complementary DNA): A piece of DNA lacking internal, non-codingsegments (introns) and regulatory sequences which determinetranscription. cDNA may be synthesized in the laboratory by reversetranscription from messenger RNA extracted from cells.

Control Cells: Cells which have not been exposed to biologicallysignificant levels of ionizing radiation.

DNA: Deoxyribonucleic acid. DNA is a long chain polymer which comprisesthe genetic material of most living organisms (some viruses have genescomprising ribonucleic acid, RNA). The repeating units in DNA polymersare four different nucleotides, each of which comprises one of the fourbases, adenine, guanine, cytosine and thymine bound to a deoxyribosesugar to which a phosphate group is attached. Triplets of nucleotides,referred to as codons, in DNA molecules code for amino acid in apolypeptide. The term codon is also used for the corresponding (andcomplementary) sequences of three nucleotides in the mRNA into which theDNA sequence is transcribed.

DNA chip: A DNA array in which multiple DNA molecules (such as cDNAs) ofknown DNA sequences are arrayed on a substrate, usually in a microarray,so that the DNA molecules can hybridize with nucleic acids (such as cDNAor RNA) from a specimen of interest. DNA chips are further described inRamsay (Nature Biotech. 16:40-44, 1998).

Differential expression of a gene: This refers to either an increased ordecreased expression of a gene, or any other change from the normalexpression of a gene.

EST (Expressed Sequence Tag): This refers to a partial DNA or cDNAsequence, typically of between 1000 and 2000 sequential nucleotides,obtained from a genomic or cDNA library, prepared from a selected cell,cell type, tissue or tissue type, organ or organism, which correspondsto an mRNA of a gene found in that library. An EST is generally a DNAmolecule sequenced from and shorter than the cDNA from which it isobtained.

Fluorophore: A chemical compound, which when excited by exposure to aparticular wavelength of light, emits light (i.e., fluoresces), forexample at a different wavelength. Fluorophores can be described interms of their emission profile, or “color.” Green fluorophores, forexample Cy-3, FITC, and Oregon Green, are characterized by theiremission at wavelengths generally in the range of 515-540 λ. Redfluorophores, for example Texas Red, Cy-5 and tetramethylrhodamine, arecharacterized by their emission at wavelengths generally in the range of590-690 λ.

Examples of fluorophores that may be used in the present invention areprovided in U.S. Pat. No. 5,866,366 to Nazarenko et al.:4-acetamido-4′-isothiocyanatostilbene-2,2′disulfonic acid, acridine andderivatives such as acridine and acridine isothiocyanate,5-(2′-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS),4-amino-N-[3-vinylsulfonyl)phenyl]naphthalimide-3,5 disulfonate (LuciferYellow VS), N-(4-anilino-1-naphthyl)maleimide, anthranilamide, BrilliantYellow, coumarin and derivatives such as coumarin,7-amino-4-methylcoumarin (AMC, Coumarin 120),7-amino-4-trifluoromethylcouluarin (Coumaran 151); cyanosine;4′,6-diaminidino-2-phenylindole (DAPI);5′,5″-dibromopyrogallol-sulfonephthalein (Bromopyrogallol Red);7-diethylamino-3-(4′-isothiocyanatophenyl)-4-methylcoumarin;diethylenetriamine pentaacetate;4,4′-diisothiocyanatodihydro-stilbene-2,2′-disulfonic acid;4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid;5-[dimethylamino]naphthalene-1-sulfonyl chloride (DNS, dansyl chloride);4-(4′-dimethylaminophenylazo)benzoic acid (DABCYL);4-dimethylaminophenylazophenyl-4′-isothiocyanate (DABITC); eosin andderivatives such as eosin and eosin isothiocyanate; erythrosin andderivatives such as erythrosin B and erythrosin isothiocyanate;ethidium; fluorescein and derivatives such as 5-carboxyfluorescein(FAM), 5-(4,6-dichlorotriazin-2-yl)aminofluorescein (DTAF),2′7′-dimethoxy-4′5′-dichloro-6-carboxyfluorescein (JOE), fluorescein,fluorescein isothiocyanate (FITC), and QFITC (XRITC); fluorescamine;IR144; IR1446; Malachite Green isothiocyanate; 4-methylumbelliferone;ortho cresolphthalein; nitrotyrosine; pararosaniline; Phenol Red;B-phycoerythrin; o-phthaldialdehyde; pyrene and derivatives such aspyrene, pyrene butyrate and succinimidyl 1-pyrene butyrate; Reactive Red4 (Cibacron® Brilliant Red 3B-A); rhodamine and derivatives such as6-carboxy-X-rhodamine (ROX), 6-carboxyrhodamine (R6G), lissaminerhodamine B sulfonyl chloride, rhodamine (Rhod), rhodamine B, rhodamine123, rhodamine X isothiocyanate, sulforhodamine B, sulforhodamine 101and sulfonyl chloride derivative of sulforhodamine 101 (Texas Red);N,N,N′,N′-tetramethyl-6-carboxyrhodamine (TAMRA); tetramethyl rhodamine;tetramethyl rhodamine isothiocyanate (TRITC); riboflavin; rosolic acidand terbium chelate derivatives.

Other suitable fluorophores include GFP, Lissamine™,diethylaminocoumarin, fluorescein chlorotriazinyl, naphthofluorescein,4,7-dichlororhodamine and xanthene and derivatives thereof. Otherfluorophores known to those skilled in the art may also be used.

Gene expression microarrays: Microscopic arrays of cDNAs printed on asubstrate, which serve as a high density hybridization target for mRNAprobes, for example as described in Schena (BioEssays 18:427-431, 1996).

Genomic target sequence: A sequence of nucleotides located in aparticular region in the human genome that corresponds to one or morespecific loci.

High throughput genomics: This refers to application of genomic orgenetic data or analysis techniques that use microarrays or othergenomic technologies to rapidly identify large numbers of genes orproteins, or distinguish their structure, expression or function fromnormal or abnormal cells or tissues.

Human Cells: Cells obtained from Homo sapiens. The cells can be obtainedfrom any source, for example peripheral blood, urine, saliva, tissuebiopsy, surgical specimen, amniocentesis samples and autopsy material.From these cells, genomic DNA, cDNA, mRNA, RNA, or protein can beisolated.

Ionizing radiation (IR): An amount of radiation sufficient to separateorbiting electrons from an atomic nucleus. Ionizing radiation includesphotons and accelerated particles. Photons (also called gamma rays) aregiven off in many types of nuclear decay. Ionizing rays (x-rays) occurwhen an electron is stopped in a dense material. Accelerated particlesinclude protons from solar radiation, heavy nuclei in cosmic rays, andbeta and alpha particles given up in nuclear decay.

Isolated: An “isolated” biological component (such as a nucleic acid,peptide, protein, or organelle) has been substantially separated,produced apart from, or purified away from other biological componentsin the cell of the organism in which the component naturally occurs,i.e., other chromosomal and extrachromosomal DNA and RNA, organelles,and proteins. Nucleic acids, peptides and proteins which have been“isolated” thus include nucleic acids and proteins purified by standardpurification methods. The term also embraces nucleic acids, peptides andproteins prepared by recombinant expression in a host cell as well aschemically synthesized nucleic acids.

Label: Detectable marker or reporter molecules, which can be attached tonucleic acids, for example probes. Typical labels include fluorophores,radioactive isotopes, ligands, chemiluminescent agents, and enzymes.Methods for labeling and guidance in the choice of labels appropriatefor various purposes are discussed, e.g., in Sambrook et al., inMolecular Cloning: A Laboratory Manual, Cold Spring Harbor LaboratoryPress (1989) and Ausubel et al., in Current Protocols in MolecularBiology, Greene Publishing Associates and Wiley-Intersciences (1987).

Malignant: cells which have the properties of anaplasia, invasion andmetastasis.

Messenger RNA (mRNA): RNA that is translated into protein. cDNA can bereverse transcribed from mRNA using standard molecular biology methods.

Microarray: An array that is miniaturized so as to require microscopicexamination for visual evaluation.

Microbes: Any microorganism. Includes for example, viruses and membersof the Monera, Protista or Fungi Kingdoms, for example bacteria andparasites.

Neoplasm: abnormal growth of cells

Normal cells: Non-tumor, non-malignant cells.

Nucleic acid array: An arrangement of nucleic acids (such as DNA or RNA)in assigned locations on a matrix, such as that found in cDNA or CGHarrays.

Nucleic acid: A deoxyribonucleotide or ribonucleotide polymer in eithersingle or double stranded form, including known analogs of naturalnucleotides.

Nucleic acid molecules representing genes: Any nucleic acid, for exampleDNA, cDNA or RNA, of any length suitable for use as a probe that isinformative about the genes.

Oligonucleotide: A linear single-stranded polynucleotide sequenceranging in length from 2 to about 1,000,000 bases, for example apolynucleotide (such as DNA or RNA) which is at least 6 nucleotides, forexample at least 15, 50, 100, 200, 1,000, 10,000 or even 1,000,000nucleotides long. Oligonucleotides are often synthetic but can also beproduced from naturally occurring polynucleotides.

Plant Cells: Cells obtained from any member of the Plantae Kingdom, acategory which includes, for example, trees, flowering and non floweringplants, grasses, and Arabidopsis. The cells can be obtained from anypart of the plant, for example roots, leaves, stems, or any flower part.From these cells, nucleic acid or protein can be isolated.

Probes and primers: A probe is an oligonucleotide or isolated nucleicacid capable of binding to a target nucleic acid of complementarysequence through one or more types of chemical bonds, usually throughcomplementary base pairing with hydrogen bond formation. Oligonucleotideprobes are often 10-50 or 15-30 bases long, and can be as long as about1,000,000 bases. An oligonucleotide probe may include natural (A, T, C,G) or modified bases (7-deazaguanosine, inosine, etc.). In addition, thebases in an oligonucleotide probe may be joined by a linkage other thana phosphodiester bond, so long as it does not interfere withhybridization. Thus, oligonucleotide probes may be peptide nucleic acidsin which the constituent bases are joined by peptide bonds rather thanphosphodiester linkages.

Detectable label or reporter molecules can be attached to probes.Typical labels include fluorophores, radioactive isotopes, ligands,chemiluminescent agents, and enzymes. Methods for labeling and guidancein the choice of labels appropriate for various purposes are discussed,e.g., in Sambrook et al., in Molecular Cloning: A Laboratory Manual,Cold Spring Harbor Laboratory Press (1989) and Ausubel et al., inCurrent Protocols in Molecular Biology, Greene Publishing Associates andWiley-Intersciences (1987).

Primers are short nucleic acids, such as DNA oligonucleotides 15nucleotides or more in length. Primers may be annealed to acomplementary target DNA strand by nucleic acid hybridization to form ahybrid between the primer and the target DNA strand, and then extendedalong the target DNA strand by a DNA polymerase enzyme. Primer pairs canbe used for amplification of a nucleic acid sequence, e.g., by thepolymerase chain reaction (PCR) or other nucleic-acid amplificationmethods known in the art.

Methods for preparing and using probes and primers are described, forexample, in Sambrook et al. (Molecular Cloning: A Laboratory Manual,Cold Spring Harbor Laboratory Press, 1989), Ausubel et al., in CurrentProtocols in Molecular Biology, Greene Publishing Associates andWiley-Intersciences (1987), and Innis et al., PCR Protocols, A Guide toMethods and Applications, 1990, Innis et al. (eds.), 21-27, AcademicPress, Inc., San Diego, Calif. PCR primer pairs can be derived from aknown sequence, for example, by using computer programs intended forthat purpose such as Primer (Version 0.5, © 11991, Whitehead Institutefor Biomedical Research, Cambridge, Mass.). One of skill in the art willappreciate that the specificity of a particular probe or primerincreases with its length. Thus, for example, a primer comprising 20consecutive nucleotides of a cDNA or gene will anneal to a targetsequence contained within a cDNA or genomic DNA library with a higherspecificity than a corresponding primer of only 15 nucleotides. Thus, inorder to obtain greater specificity, probes and primers may be selectedthat comprise 20, 25, 30, 35, 40, 50 or more consecutive nucleotides ofa cDNA or gene sequence.

Thus isolated nucleic acid molecules that comprise specified lengths ofa nucleic acid sequence can be used in the present invention. Suchmolecules may comprise at least 20, 40, 50, 100, 1000, 10,000, or even1,000,000 or more consecutive nucleotides of a nucleic acid sequence andmay be obtained from any region of a nucleic acid sequence.

Probe Element: A nucleic acid sequence from a gene which is representedin a probe set. For example, the gene may be differentially expressed bya cell following radiation induced biological stress. In addition, thegene may be unaffected in its expression following radiation inducedbiological stress, for example control genes.

Probe set: A population of two or more probes which represent genes thatare differentially expressed in cells that have been exposed to abiologically significant amount of ionizing radiation. The probe set maybe nucleic acids, for example RNA, cDNAs, and/or oligonucleotides thatspecifically hybridize to complementary sequences in a nucleic acidcomposition, for example from test cells which may have been exposed toionizing radiation. For example, the probe set may contain any of theprobes shown in Tables 4 or 5. In addition, the probe set can be boundin an array to a surface.

Purified: the term purified does not require absolute purity; rather, itis intended as a relative term. Thus, for example, a purified proteinpreparation is one in which the protein is more pure than the protein inits natural environment within a cell. In one embodiment, a preparationof a protein is purified such that the protein represents at least 50%of the total protein content of the preparation.

Radiation dose: Defined in terms of energy deposition. The basic unit isthe gray (Gy), equal to 1 joule per kilogram.

Radiation induced biological stress: The induction of differentialexpression of stress genes in cells after they are exposed to radiation.Examples include, but are not limited to: CIP1/WAF1, GADD45, MDM2, BCL2,FOS, JUN, REL-B, ATF3, BAX.

Sequence identity: The similarity between two nucleic acid sequences, ortwo amino acid sequences, is expressed in terms of the similaritybetween the sequences, otherwise referred to as sequence identity.Sequence identity is frequently measured in terms of percentage identity(or similarity or homology); the higher the percentage, the more similarthe two sequences are. Homologs or orthologs of nucleic acid or aminoacid sequences will possess a relatively high degree of sequenceidentity when aligned using standard methods. This homology will be moresignificant when the orthologous proteins or nucleic acids are derivedfrom species which are more closely related (e.g., human and chimpanzeesequences), compared to species more distantly related (e.g., human andC. elegans sequences). Typically, orthologs are at least 50% identicalat the nucleotide level and at least 50% identical at the amino acidlevel when comparing human orthologous sequences.

Methods of alignment of sequences for comparison are well known in theart. Various programs and alignment algorithms are described in: Smith &Waterman, Adv. Appl. Math. 2:482, 1981; Needleman & Wunsch, J. Mol.Biol. 48:443, 1970; Pearson & Lipman, Proc. Natl. Acad. Sci. USA85:2444, 1988; Higgins & Sharp, Gene, 73:237-44, 1988; Higgins & Sharp,CABIOS 5:151-3, 1989; Corpet et al., Nuc. Acids Res. 16:10881-90, 1988;Huang et al. Computer Appls. in the Biosciences 8, 155-65, 1992; andPearson et al., Meth. Mol. Bio. 24:307-31, 1994. Altschul et al., J.Mol. Biol. 215:403-10, 1990, presents a detailed consideration ofsequence alignment methods and homology calculations.

The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al. J.Mol. Biol. 215:403-10, 1990) is available from several sources,including the National Center for Biotechnology Information (NCBI,Bethesda, Md.) and on the Internet, for use in connection with thesequence analysis programs blastp, blastn, blastx, tblastn and tblastx.A description of how to determine sequence identity using this programis available at the NCBI website.

Homologs are typically characterized by possession of at least 60%, 70%,75%, 80%, 90%, 95% or at least 98% sequence identity counted over thefull length alignment with a sequence using the NCBI Blast 2.0, gappedblastp set to default parameters. Queries searched with the blastnprogram are filtered with DUST (Hancock, and Armstrong, 1994, Comput.Appl. Biosci. 10:67-70). Other programs use SEG.

One of ordinary skill in the art will appreciate that these sequenceidentity ranges are provided for guidance only; it is entirely possiblethat strongly significant homologs could be obtained that fall outsideof the ranges provided. The present invention provides not only thepeptide homologs described above, but also nucleic acid molecules thatencode such homologs.

One indication that two nucleic acid sequences are substantiallyidentical is that the polypeptide which the first nucleic acid encodesis immunologically cross reactive with the polypeptide encoded by thesecond nucleic acid.

Nucleic acid sequences that do not show a high degree of identity maynevertheless encode similar amino acid sequences, due to the degeneracyof the genetic code. It is understood that changes in nucleic acidsequence can be made using this degeneracy to produce multiple nucleicacid sequences that all encode substantially the same protein.

An alternative indication that two nucleic acid molecules are closelyrelated is that the two molecules hybridize to each other understringent conditions, as described under “specific hybridization.”

Specific hybridization: Specific hybridization refers to the binding,duplexing, or hybridizing of a molecule only or substantially only to aparticular nucleotide sequence when that sequence is present in acomplex mixture (e.g. total cellular DNA or RNA). Specific hybridizationmay also occur under conditions of varying stringency.

Hybridization conditions resulting in particular degrees of stringencywill vary depending upon the nature of the hybridization method ofchoice and the composition and length of the hybridizing DNA used.Generally, the temperature of hybridization and the ionic strength(especially the Na⁺ concentration) of the hybridization buffer willdetermine the stringency of hybridization. Calculations regardinghybridization conditions required for attaining particular degrees ofstringency are discussed by Sambrook et al. (In: Molecular Cloning: ALaboratory Manual, Cold Spring Harbor, N.Y., 1989 ch. 9 and 11). By wayof illustration only, a hybridization experiment may be performed byhybridization of a DNA molecule to a target DNA molecule which has beenelectrophoresed in an agarose gel and transferred to a nitrocellulosemembrane by Southern blotting (Southern, J. Mol. Biol. 98:503, 1975), atechnique well known in the art and described in Sambrook et al.(Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, N.Y.,1989).

Hybridization with a target probe labeled with [³²P]-dCTP is generallycarried out in a solution of high ionic strength such as 6×SSC at atemperature that is 20-25° C. below the melting temperature, T_(m),described below. For such Southern hybridization experiments where thetarget DNA molecule on the Southern blot contains 10 ng of DNA or more,hybridization is typically carried out for 6-8 hours using 1-2 ng/mlradiolabeled probe (of specific activity equal to 10⁹ CPM/μg orgreater). Following hybridization, the nitrocellulose filter is washedto remove background hybridization. The washing conditions should be asstringent as possible to remove background hybridization but to retain aspecific hybridization signal.

The term T_(m) represents the temperature (under defined ionic strength,pH and nucleic acid concentration) at which 50% of the probescomplementary to the target sequence hybridize to the target sequence atequilibrium. Because the target sequences are generally present inexcess, at T_(m) 50% of the probes are occupied at equilibrium. TheT_(m) of such a hybrid molecule may be estimated from the followingequation (Bolton and McCarthy, Proc. Natl. Acad. Sci. USA 48:1390,1962): T_(m)=81.5° C.−16.6(log₁₀[Na⁺])+0.41(% G+C)−0.63(%formamide)−(600/l); where l=the length of the hybrid in base pairs.

This equation is valid for concentrations of Na⁺ in the range of 0.01 Mto 0.4 M, and it is less accurate for calculations of T_(m) in solutionsof higher [Na⁺]. The equation is also primarily valid for DNAs whose G+Ccontent is in the range of 30% to 75%, and it applies to hybrids greaterthan 100 nucleotides in length (the behavior of oligonucleotide probesis described in detail in Ch. 11 of Sambrook et al. (Molecular Cloning:A Laboratory Manual, Cold Spring Harbor, N.Y., 1989).

Thus, by way of example, for a 150 base pair DNA probe derived from acDNA (with a hypothetical % GC=45%), a calculation of hybridizationconditions required to give particular stringencies may be made asfollows: For this example, it is assumed that the filter will be washedin 0.3×SSC solution following hybridization, thereby: [Na⁺]=0.045 M; %GC=45%; Formamide concentration=0; 1=150 base pairs;T_(m)=81.5−16.6(log₁₀[Na⁺])+(0.41×45)−(600/150); and so T_(m)=74.4° C.

The T_(m) of double-stranded DNA decreases by 1-1.5° C. with every 1%decrease in homology (Bonner et al., J. Mol. Biol. 81:123, 1973).Therefore, for this given example, washing the filter in 0.3×SSC at59.4-64.4° C. will produce a stringency of hybridization equivalent to90%; that is, DNA molecules with more than 10% sequence variationrelative to the target cDNA will not hybridize. Alternatively, washingthe hybridized filter in 0.3×SSC at a temperature of 65.4-68.4° C. willyield a hybridization stringency of 94%; that is, DNA molecules withmore than 6% sequence variation relative to the target cDNA moleculewill not hybridize. The above example is given entirely by way oftheoretical illustration. One skilled in the art will appreciate thatother hybridization techniques may be utilized and that variations inexperimental conditions will necessitate alternative calculations forstringency.

In the present invention, stringent conditions may be defined as thoseunder which DNA molecules with more than 25%, 15%, 10%, 6% or 2%sequence variation (also termed “mismatch”) will not hybridize.Stringent conditions are sequence dependent and are different indifferent circumstances. Longer sequences hybridize specifically athigher temperatures. Generally, stringent conditions are selected to beabout 5° C. lower than the thermal melting point T_(m) for the specificsequence at a defined ionic strength and pH. An example of stringentconditions is a salt concentration of at least about 0.01 to 1.0 M Naion concentration (or other salts) at pH 7.0 to 8.3 and a temperature ofat least about 30° C. for short probes (e.g. 10 to 50 nucleotides).Stringent conditions can also be achieved with the addition ofdestabilizing agents such as formamide. For example, conditions of5×SSPE (750 mM NaCl, 50 mM Na Phosphate, 5 mM EDTA, pH 7.4) and atemperature of 25-30° C. are suitable for allele-specific probehybridizations.

A perfectly matched probe has a sequence perfectly complementary to aparticular target sequence. The test probe is typically perfectlycomplementary to a portion (subsequence) of the target sequence. Theterm “mismatch probe” refers to probes whose sequence is deliberatelyselected not to be perfectly complementary to a particular targetsequence.

Transcription levels can be quantitated absolutely or relatively.Absolute quantitation can be accomplished by inclusion of knownconcentrations of one or more target nucleic acids (for example controlnucleic acids such as Bio B or with a known amount the target nucleicacids themselves) and referencing the hybridization intensity ofunknowns with the known target nucleic acids (for example by generationof a standard curve).

Subject: Living multicellular vertebrate organisms, a category whichincludes, both human and veterinary subjects for example, mammals, birdsand primates.

Test Cell: A cell which has been or may have been exposed tobiologically significant levels of ionizing radiation. Using the methodof the present invention, whether a test cell has been exposed tobiologically significant levels of ionizing radiation can be determined.Test cells can be from any origin, including for example plant andanimal cells. In particular examples, the test cells are peripheralblood cells.

Tumor: a neoplasm

Additional definitions of terms commonly used in molecular genetics canbe found in Benjamin Lewin, Genes V published by Oxford UniversityPress, 1994 (ISBN 0-19-854287-9); Kendrew et al (eds.), The Encyclopediaof Molecular Biology, published by Blackwell Science Ltd., 1994 (ISBN0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biology andBiotechnology: a Comprehensive Desk Reference, published by VCHPublishers, Inc., 1995 (ISBN 1-56081-569-8).

EXAMPLE 1 Apoptosis of ML Cells after Irradiation

This example describes experiments in which the percent of cellsundergoing apoptosis following different levels of irradiation wascalculated.

ML-1 cells, a human myeloid leukemia cell line, were grown in RPMI 1640medium supplemented with 10% heat-inactivated (56° C. for 45 minutes)fetal calf serum and 100 U/ml penicillin and 100 μg/ml streptomycin in ahumidified, 5% CO₂ atmosphere in a 37° incubator. Cells were irradiatedat approximately 5.1 cGy/minute to total doses between 2 and 2000 cGyusing a Mark 1-68 ¹³⁷Cs source (J.L. Shepherd and Associates, Inc., SanFernando, Calif.) with lead attenuators in place. The dosimetry of thesource was confirmed by exposing TLD monitors (Landauer Inc., Glenwood,Ill.) in the same configuration used for cellular irradiations to therange of doses used. Even at the lowest doses, the calculated absorbeddose (Landauer special dosimetry services) varied by less than 3% fromthe dose expected. Due to the nature of sparsely ionizing radiation suchas γ-rays, it is highly unlikely that cells in the irradiated populationwill remain unexposed at even the lowest doses used.

To determine if cells underwent apoptosis following irradiation, thecells were irradiated and then incubated for 1, 2, or 3 days. The cellswere subsequently fixed in methanol, and stained with DAPI solution (50ng/ml final concentration). An Olympus fluorescent microscope was usedto score nuclei exhibiting characteristic morphological features ofapoptosis, and results were expressed as the number of apoptotic nucleiover the total number of nuclei counted. Flow cytometry was used tomeasure the effects of irradiation on the cell cycle. In this method,cells were fixed in 70% ethanol 0, 8, 10, 12 and 24 hours afterirradiation, treated with RNase (100 ug/ml) at 37° C., then stained withpropidium iodide. Samples were analyzed using a Becton-Dickenson FacScanand cell cycle distributions were fitted using the Cell Quest dataanalysis program.

ML-1 cells irradiated with ¹³⁷Cs γ-rays showed a survival responsesimilar to that of other human cell lines of myeloid or lymphoid lineage(FIG. 1), which are typically more radiosensitive than cell linesderived from other tissue types. The survival curve had no apparentshoulder and predicted a D₀ of approximately 73 cGy. The changes inplating efficiency induced by doses of 2, 5, and 10 cGy were so low asnot to be measurably different from fluctuations in the colony formationassay (p>0.1). In four independent experiments, the mean platingefficiency of untreated controls was 0.129+/−0.069 while the meanplating efficiency for all doses combined (2, 5 and 10 cGy) was0.127+/−0.058.

Although previous studies demonstrated that high doses of ionizingradiation efficiently induce rapid apoptosis in ML-1 cells (Zhan et al.Oncogene 9:3743-51, 1994), at lower, relatively non-toxic doses (cellsirradiated with 25 cGy or less), very little apoptosis was measurable. Amore sensitive method for detecting apoptotic cells was achieved byscoring morphology of DAPI stained cells. As shown in FIG. 2, two daysafter irradiation, a trend of increasing apoptosis with increasing doseemerged between 2 and 25 cGy, although due to experimental fluctuations,this increase was only significant at the p<0.01 level (paired t-test)following the 25 cGy dose. In contrast, cultures treated with 250 cGyshowed over 40% morphologically apoptotic cells two days afterirradiation. No further increases in apoptotic fraction were observed attimes later than two days following treatment for any of the dosestested.

In contrast to their effect on apoptosis, doses below 25 cGysignificantly and reproducibly perturbed cell cycle progression in adose-dependent manner (FIGS. 3A and 3B). Even the lowest dose tested, 2cGy, caused a transient reduction of the S-phase fraction. This effectwas only significant at 12 hours after irradiation with 2 cGy, whileincreased doses resulted in larger and more rapid decreases in theS-phase fraction of the population (FIGS. 3A and 3B). By 24 hourspost-irradiation, the populations treated with doses of 25 cGy or belowhad all recovered their normal cell cycle distributions, while cellstreated with the higher dose of 250 cGy still showed a profoundlyarrested profile with very few (6.6+/−1.9 percent) S-phase cells.

These results demonstrate that low-levels of non-toxic irradiation canbe effectively administered, and that even at these low doses,progression of the cell cycle is perturbed.

EXAMPLE 2 Construction of cDNA Microarrays

This example describes experiments conducted to identify genes which aredifferentially expressed in cells exposed to radiation, such as lowlevel radiation, which were used to construct cDNA microarrays. Before aprobe set can be constructed which identifies differential expression ofgenes in radiation exposed cells, probes are selected for the set. Thisselection can be achieved by using cDNA arrays having a general samplingof ESTs, for example from human, mouse, or plant genes. The array ofESTs can be exposed to nucleic acid compositions from test cells thathave been exposed to a dose of ionizing radiation sufficient to inducedifferential expression of stress genes in the test cells. The nucleicacids can then be labeled (for example by reverse transcription fromcellular mRNA to labeled cDNA), and exposed to the array. Specifichybridization to an EST in the array, or differential hybridization by acDNA from a cell that has been irradiated, identifies a potential probefor the probe set. Subsequent confirmation of differential expressioncan be achieved by northern blot analysis, or other techniques.

The microarray used in this example included a general sampling of humangenes (622 ESTs) plus another set of genes (616 ESTs) which were chosenon the basis of their roles in cancer or lymphoid biology. The genesrepresented in the 1.2K array are shown in Table 1. The sequences areidentified in Table 1 by Image Consortium Clone number (hereinafter“Image No.”). This number can be searched on the ATCC Image ConsortiumClones database at the ATCC (Manassas, Va.) website, with links to theATCC accession number of clones from which the sequences can beobtained, and links to Genbank sequence listings which correspond to theImage Number. The array was constructed with ESTs informative for theexposure of interest, namely radiation exposure. A panel of housekeepinggenes and other internal controls was also included on the array. Theselection of this panel has been described (DeRisi et al., Nat. Genet.14:457-460, 1996). TABLE 1 Image ID Numbers of Genes Present in the 1.2Karray. 21420; 22036; 29054; 33941; 34327; 38805; 42894; 43110; 47359;471252; 486375; 80549; 161023; 51363; 310406; 470470; 485963; 298371;62186; 41959; 25081; 25725; 32198; 36593; 36844; 40765; 44527; 44710;49888; 51528; 51740; 31116; 253773; 266486; 364278; 51825; 193913;363805; 344482; 360201; 21477; 22040; 29204; 34011; 34357; 38829; 42906;43129; 47384; 471494; 486233; 81221; 162584; 51439; 310490; 470685;486161; 298371; 80549; 135118; 25154; 25764; 32304; 36676; 36904; 40831;44537; 44721; 49897; 51540; 51785; 32790; 253869; 267246; 364982; 52193;196543; 364110; 344769; 361401; 21468; 22230; 29234; 34005; 34349;38840; 42910; 43194; 47386; 471715; 486086; 82991; 162772; 51746;310390; 470730; 486074; 299626; 110589; 136769; 25169; 25880; 32319;36681; 36916; 40874; 44563; 44722; 49898; 51555; 51814; 32983; 257766;267682; 364752; 52564; 199381; 364975; 345527; 361815; 21531; 22260;29435; 34141; 34355; 38859; 42927; 43229; 47463; 472067; 486785; 82850;48398; 51854; 310428; 470691; 486716; 300051; 113941; 154763; 25400;26052; 32409; 36700; 36922; 40886; 44584; 45188; 49926; 51621; 51940;33368; 258589; 268652; 364716; 178169; 200209; 366539; 347573; 366981;21652; 28573; 29438; 34217; 38567; 38876; 42993; 47229; 47475; 484993;62186; 84730; 49164; 307741; 322000; 471217; 296021; 301363; 121147;25485; 31852; 32407; 36717; 40493; 40911; 44605; 49751; 49967; 51666;27944; 34005; 260052; 362009; 365147; 178508; 361692; 366509; 347685;21656; 28595; 29627; 34247; 38573; 38966; 43021; 47306; 47493; 485690;77577; 85906; 49950; 309615; 322604; 471620; 296642; 303109; 129268;25495; 31855; 32438; 36775; 40562; 40959; 44666; 49777; 49980; 51685;28573; 35326; 261518; 362332; 365647; 182633; 361692; 366942; 357357;22012; 28776; 29629; 34255; 38647; 38983; 43060; 47343; 47499; 486012;79742; 86017; 50576; 309477; 323162; 471822; 297589; 306170; 26503;25517; 31988; 32427; 36796; 40670; 40991; 44689; 49788; 50038; 51687;28774; 35516; 262575; 362926; 366420; 183661; 362702; 375620; 358677;21810; 28985; 29640; 34304; 38770; 39086; 43092; 47333; 47631; 486335;80239; 23464; 50503; 310141; 323001; 471918; 298187; 307471; 26599;25584; 32141; 32531; 36790; 40764; 41094; 44699; 49849; 50032; 51699;30781; 36074; 264606; 363799; 366576; 191007; 363539; 375635; 358538;22285; 22589; 30543; 34396; 34671; 39173; 43231; 43563; 47833; 486560;488373; 110589; 52489; 193736; 323776; 486510; 489217; 321246; 162479;231292; 26082; 26483; 32649; 36927; 37230; 41439; 45222; 45643; 50232;51961; 52587; 43129; 268727; 279482; 375922; 205905; 236420; 377275;375635; 417127; 22293; 22611; 30573; 34537; 34761; 39195; 43338; 43601;47874; 486844; 488801; 110503; 52162; 190711; 324698; 487130; 489194;321308; 50576; 247869; 26087; 26531; 32644; 36950; 37231; 41452; 45233;45658; 50234; 52019; 52629; 131828; 270148; 279954; 376362; 209655;239287; 377181; 375724; 418320; 22379; 22798; 30578; 34555; 34860;39205; 43241; 43622; 47884; 487417; 488989; 112035; 52681; 200378;324655; 487097; 510130; 322148; 52681; 252185; 26129; 26541; 32658;36983; 37234; 41511; 45291; 45794; 50271; 52222; 52650; 132420; 270980;281029; 376285; 213502; 241489; 377441; 375843; 428248; 22445; 22790;30588; 34606; 34945; 39270; 43409; 43631; 48077; 487264; 489419; 114116;179711; 201797; 324815; 487777; 510258; 322452; 190107; 256323; 26128;26572; 32682; 36975; 37269; 41559; 45376; 45851; 50308; 52342; 52652;133122; 271478; 281778; 376737; 219735; 242134; 415206; 376294; 470792;22493; 29799; 30592; 34637; 39093; 39285; 43504; 47648; 48085; 487386;108284; 114648; 180447; 323028; 325182; 487965; 308392; 323181; 192694;26147; 32609; 32684; 37040; 41208; 41773; 45421; 50117; 50322; 52338;36215; 135381; 271416; 366647; 376649; 231089; 376110; 415060; 376218;22545; 30012; 30622; 34647; 39148; 39380; 43541; 47679; 48097; 487878;108837; 115277; 183950; 323390; 327396; 488891; 320514; 323648; 193901;26184; 32615; 32717; 37118; 41199; 41796; 45564; 50106; 50359; 52415;36844; 137531; 271662; 366824; 380403; 231292; 376303; 415636; 376507;22568; 30149; 30619; 34662; 39159; 39391; 43549; 47727; 48234; 488103;110022; 115408; 187987; 323555; 328692; 489042; 320903; 324700; 209310;26286; 32626; 32707; 37145; 41356; 41792; 45568; 50171; 50363; 52489;39920; 137575; 272110; 366904; 415102; 231497; 376781; 415679; 380738;22589; 30272; 30664; 34660; 39169; 39542; 43550; 47838; 48406; 488596;109957; 115336; 192694; 323500; 340644; 489327; 321242; 328330; 222502;26366; 32636; 32750; 37196; 41392; 41813; 45604; 50203; 50503; 52530;41565; 138460; 278409; 375724; 415731; 232714; 376725; 416184; 381219;23014; 23731; 30885; 34974; 35609; 39827; 43711; 44105; 48677; 489395;510245; 121849; 201673; 221690; 343166; 510467; 112500; 343465; 257458;282065; 26585; 27473; 32898; 37347; 37508; 42059; 45840; 46329; 50603;52681; 53084; 140358; 283116; 293274; 417403; 243143; 262691; 427923;471218; 488658; 23019; 23804; 30970; 35110; 35623; 39861; 43719; 44159;48717; 509710; 512458; 123586; 203132; 221619; 343871; 512391; 120466;343311; 260288; 286197; 26593; 27511; 33008; 37366; 37513; 42088; 45941;46339; 50666; 52753; 53107; 140827; 284031; 293934; 417592; 253545;264576; 427943; 471631; 488904; 23073; 23831; 30966; 35284; 35615;39876; 43826; 44173; 48713; 509731; 512472; 123953; 203017; 223176;344109; 198205; 124086; 343646; 264200; 288650; 26659; 27549; 33045;37380; 37622; 42096; 46019; 46354; 50816; 52773; 53128; 143751; 284459;295208; 417689; 254428; 264554; 427943; 471889; 509710; 23132; 23866;30981; 35297; 35665; 39884; 43858; 44180; 48762; 509820; 512385; 124554;204301; 230976; 345600; 322029; 26992; 345645; 271416; 290117; 26789;27624; 33183; 37435; 37679; 42130; 46042; 46419; 50888; 52896; 53155;144944; 288797; 298314; 418111; 254187; 268412; 427750; 485192; 509682;23185; 30691; 31061; 35313; 39686; 39903; 43885; 48452; 48803; 510101;116906; 125187; 204681; 340734; 345928; 60298; 328467; 345722; 272110;26801; 32774; 33176; 37433; 41857; 42214; 46171; 50555; 50941; 52931;138604; 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42888; 46936; 51293; 51504; 53316; 152844; 159833;305455; 429976; 470621; 289428; 469731; 471174; 195889

The human myeloid leukemia cell line ML-1 is available from the NationalCancer Institute's Anti-Neoplastic Drug Screen Panel (NCI-ADS). Themembers of this panel are listed in Table 2, and are described in manypublications, including Monks et al. (J. Nat. Cancer Institut.83:757-66, 1991), and on the NCI Web page. ML-1 was selected as the testcell in which radiation induced differential expression of genes wouldbe measured. A characteristic of this cell that made it particularlyuseful for this purpose was that it contains endogenous wild-type p53,which is a cellular stress response mediator following exposure toionizing radiation. ML-1 contains physiologic levels of p53, rather thanthe unnatural and often highly overexpressed levels often seen inartificially engineered systems. Moreover, ML-1 is a myeloid cell line,which is prone to undergo rapid apoptosis following genotoxic stress.This characteristic results in the induction of genes specificallyassociated with rapid apoptosis, such as BAX, MCLI, GADD34 and BCL-X(Zhan et al., Oncogene 9:3743-51, 1994 and Oncogene 14:1031-9, 1997). Inaddition to ML-1 cells, other cells that can be used to assess theinduction of differential expression of genes in radiation exposed cellsinclude other lymphoid cell lines, such as those with wild-type levelsof p53, such as Molt4, WMN, TK6 or SR, which may induce genes not foundto be regulated in ML-1. Cell lines derived from blood cells (such asthese lines) are selected to provide responses more characteristic ofperipheral blood responses than would cell lines from other sites, suchas colon or breast cell lines (for example RKO or MCF7, respectively).Gene induction can also be measured directly in ex vivo irradiated humanperipheral blood cells, as a particularly relevant model system, formethods in which peripheral blood specimens are analyzed to clinicallyscreen for biologically significant radiation exposure (see EXAMPLE 9).TABLE 2 The National Cancer Institute's Anti-Neoplastic Drug ScreenPanel* Tissue Origin Cell Line Names Breast Cancer BT-549; HS 578T;MCF7; MDA-MB-231; MDA-MB435; MDA-N; NCI-ADR; T-47D; NCI/ADR-RES; MAXF401; MDA-MB-468; SK-BR-3 CNS Cancer SF-268; SF-295; SF-539; SNB-19;SNB-75; U251; SNB-78; TE671; XF 498 Colon Cancer COLO205; HCC-2998;HCT116; HCT15; HT29; KM12; RKO; SW-620; DLD-1; KM20L2 Non Small CellA549; EKVX; HOP-62; HOP-92; NCI-H226; NCI-H23; NCI-H322M; Lung CancerNCI-H460; NCI-H522; HOP-18; HOP-19; LXFL 529 Small Cell Lung DMS 114;DMS 273; SHP-77 Cancer Leukemia CCRF-CEM; HL60; K562; MOLT4; RPMI-8226;SR; P388 P388/ADR Melanoma LOX-IVMI; M14; MALME-3M; SK-MEL-2; SK-MEL-28;SK-MEL-5; UACC-257; UACC-62; RPMI-7951; M19-MEL Ovarian Cancer IGROV-1;OVCAR-3; OVCAR-4; OVCAR-5; OVCAR-8; SK-OV-3 Prostate Cancer DU-145; PC-3Renal Cancer 786-O; A498; ACHN; CAKI-1; RXF 393; SN12C; TK-10; UO-31;RXF-631; SN12K1*Cells are available either from ATCC (Manassas, VA) or from theFrederick cancer research facility in Frederick, MD.

ML-1 cells were grown and irradiated at approximately 3.1 Gy/minute tototal doses of 0.25, 2 or 20 Gy using a Mark 1-68 ¹³⁷CS source, asdescribed in EXAMPLE 1. EST targets identified in Table 1 were preparedby PCR amplification and arrayed on poly-L-lysine coated glass slides byhigh speed robotic printing as previously described (DeRisi et al., Nat.Genet. 14:457-460, 1996).

A complex cDNA probe was prepared from whole-cell RNA by a single roundof reverse transcription using SuperscriptII (Gibco-BRL, Grand Island,N.Y.) for two hours at 42° C. according to the manufacturer'sinstructions in the presence of fluorescent dNTP (Cy3 dUTP or Cy5 dUTP,Amersham Pharmacia Biotech, Piscataway, N.J.). Probes were hybridized tothe slides for 16 hours in 3×SSC (8.77 g/L NaCl, 4.41 g sodium citrateat pH 7.4) at 65° C. in the presence of blockers (8 μg Poly d A; 4 μgyeast tRNA; and 10 μg human Cot 1 per 10 μl hybridization). Hybridizedslides were washed at room temperature (RT) in 0.5×SSC, 0.01% SDS, thenin 0.06×SSC. The two fluorescent intensities (Cy3 and Cy5) were scannedseparately using a laser confocal microscope, and the DeArray programwas then used to identify target sites by image segmentation, calibraterelative ratios, and to develop confidence intervals for testing thesignificance of the ratios obtained (Chen et al., J. Biomed. Optics2:364-374, 1997).

Local background was calculated for each target location. Anormalization factor was estimated from a set of 88 internal controltargets (De Risi et al., Nat. Genet, 14:457-60, 1996) with a theoreticalratio of 1.0, and the confidence interval for the array was estimatedfrom the variance of these 88 control ratios from the expected value of1.0. The ratios for all the targets on the array were then calibratedusing the normalization factor, and ratios outside the 99% confidenceinterval (less than 0.54 or greater than 2.37) were determined to besignificantly changed by the radiation treatment.

In the hybridized microarray, induced transcripts hybridized with moreof the probe from the IR-treated sample (labeled with a redfluorophore), resulting in red spots, such as that observed at theCIP1/WAF1 target. A transcript down-regulated by IR, such as c-MYC,instead produced a green spot. Intermediate induction ratios result in agradation of color, such as MBP-1. A schematic drawing showing thehybridized microarray is shown in FIG. 4, in which red spots are shownwith vertical lines, green spots are shown with horizontal lines, andyellow spots (which have an equal representation of read an green label)are represented with both horizontal and vertical lines. One skilled inthe art will appreciate that the color of the labels used is notcritical, so long as the emission wavelength of the differentfluorophores used can be resolved, and can be used to measuredifferential expression. Other fluorophores or labels can be used topractice the method of the present invention.

Transcripts significantly changed by radiation treatment are shown inTable 3 which also gives the mean intensities of hybridization to theunirradiated control on the microarray. This measure correlates withtranscript abundance (Schena et al., Science 270:467-70, 1995; Schena etal., Proc. Natl. Acad. Sci. USA 93:10614-9, 1996), and demonstratesidentification of IR modulated genes over three orders of magnitude ofbasal expression. Many of the stress regulated transcripts identified inTable 3 are known to be expressed at very low levels in ML-1 cells,consistent with their relative hybridization intensities on the array.For example, GADD45 and CIP1/WAF1 represent approximately 1/10⁵transcripts in unirradiated cells. TABLE 3 Stress gene responses inγ-irradiated human myeloid cells Mean Green Transcript Image IDMicroarray^(a) Intensity^(b) CIP1/WAF1 268652 42.0^(c) 1121 ATF3 42824811.3^(c) 784 FAS 151767 9.5 1352 IAP-1 129632 9.3^(d) 132 .RELB 526817.2^(d) 1027 cyclin I 512391 6.9 974 RAB 52530 6.4 1688 GADD45 3101415.8^(d) 2230 FRA1 110503 4.8^(c) 672 IL-8 328692 4.8 5307 CSF-1 1245544.7 356 BCL3 236422 4.6^(c) 584 MIP1α 153355 4.3 475 c-FOS 26474 4.21314 JUN-B 309477 4.2 1892 PC-1 52753 3.4^(c) 343 CDC5 280376 3.4 1259MRC OX-2 51363 3.4^(d) 290 ERR1 470602 3.2 490 MDM2 147075 3.2^(d) 328Immunoglobulin J chain 161023 3.1 331 OX40 ligand 35326 3.1 310 DNAligase III 470062 3.0 636 cytochrome p450 4A 120466 3.0 218 MEK1 4860742.9 1885 TPK receptor UFO 112500 2.8 285 Retinoic acid gamma-1 4712522.8 288 HPTP alpha 487130 2.8 282 MBP-1 291503 2.8^(c) 206 SSAT 414522.7^(c) 4160 BAK e 2.7^(c) 341 MBP-2 23464 2.5 462 iL-TMP 243143 2.5^(d)383 MIP1beta 205633 2.5 352 N-RAS 284031 2.5 341 nucleotide bindingprotein 21420 2.5 394 mRNA CAP-R 48677 2.4 577 BCL-XL e 2.3^(c) 313ch-TOG 43060 0.53 9209 ERK 487417 0.51 9323 CDC2 346534 0.47 19501 SATB1233194 0.46^(c) 30364 SPI-B 295093 0.45 3747 ERF-1 48085 0.44 6935 MADP2homolog 26541 0.43 20543 neuron-specific protein gene 28089 0.38 8098TOPO II 248032 0.34^(c) 37173 CDK RS2 359119 0.27 13935 RCH-1 281160.23^(c) 24932 c-MYC 51699 0.12^(c) 17913^(a)Ratios of relative induction by γ-rays compared to basal levels inML-1 cells.^(b)Fluorescence intensity of untreated control on the microarray.^(c)Microarray measurement confirmed by quantitative dot blothybridization where expression varied by less than 2-fold.^(d)Quantitation varied by more than 2-fold.^(e)Inserts for BAK (Chittenden et al., Nature 374: 733-6, 1995) andBCL-XL (Boise et al., Cell 74: 597-608, 1993) from clones other thanImage Consortium ESTs.

A subset of the IR-responsive genes indicated by the microarray with arange of relative ratios were chosen for further study. This subset ofprobes included the following: Image ID clones 268652, 428248, 129632,52681, 52530, 310141, 110503, 236422, 153355, 52753, 51363, 147075,291503, 41452, 243143, 233194, 248032, 28116, 51699. Probes wereobtained from the same plasmids used as targets on the array, and γ-rayinduction of these genes was confirmed in independent experiments bynorthern blot hybridization. Estimates of induction or repression asmeasured by the microarray were compared to quantitative hybridizationwith single labeled probes. As indicated in Table 3, estimatedexpression varied by less than 2-fold for many transcripts. However, alltested sequences that were identified on the microarray as inducedshowed an appreciable (>2-fold) induction by the quantitativehybridization approach, with the exception of MRC-OX, which showed only1.5-fold induction. In the case of genes showing less than 2.4-foldinduction by the microarray, useful data may still be obtainable. Forexample, MCL-1 showed 1.9-fold induction by the microarray and 2.5-foldinduction by quantitative hybridization.

The time course of induction for nine of these genes was examined inML-1 cells. The response over time of Rag cohort 1 (RCH1) (Cuomo et al.,Proc. Natl. Acad. Sci. USA 91:6156-60, 1994), a newly recognizedIR-down-regulated gene, was very similar to the response of TOPOII. Bothrepressed genes showed a similar rapid decrease of mRNA levels followingirradiation, and remain maximally repressed 24 hours after treatment.The levels of most of the newly-identified IR-induced genes rose rapidlyfollowing treatment, peaked by four hours, and declined again to nearthe original levels by 24 hours after treatment, following the patternof rapid response typical of many stress-induced immediate-early genes(Fornace, Ann. Rev. Genet. 26:507-26, 1992; Smith and Formace, MutationRes. 340:109-24, 1996). By analogy, the genes described here appear tohave roles in acute cellular responses to damage and may share someregulatory mechanisms with previously characterized IR-response genes.

Induction of nine of the newly-identified stress-response genes was nextmeasured in a panel of human cancer cell lines to determine the scope oftheir IR-response, and to monitor for induction by exposure to twoDNA-base-damaging agents, the alkylating agent methyl methanesulfonate(MMS) and ultraviolet (UV) radiation. The full list of cell lines fromthis panel is shown in Table 2.

The cell lines used in this comparison included six cell lines ofmyeloid-lymphoid lineage [ML-1 (myeloid), Molt4 (lymphoid), SR(lymphoid), CCRF-CEM (lymphoid), HL60 (myeloid) and K562 (myeloid)], twolung cancer lines (A549 and H1299), two breast carcinoma lines (MCF7 andT47D), and the colon cancer line RKO, along with its derivativetransfected with E6 (RKO/E6) (Zhan et al., Mol. Cell. Biol. 13:4242-50,1993). Relative induction of particular genes was measured four hoursafter treatment with a 20 Gy dose of ionizing radiation (FIG. 5A) or 100μg/ml MMS or 14 J/m² UV radiation (FIG. 5B) as measured by quantitativedot-blot hybridization (Hollander and Fornace, Biotechniques 9:174-9,1990), using the same conditions as described above. Differentialexpression of genes in tumor cells following radiation exposure can beused as a measure of therapeutic radiosensitivity, or to help studyexpression patterns that can be the subject of other therapeutic (e.g.drug) interventions.

A summary of the results of these induction experiments is shown inFIGS. 5A and 5B, in which induced genes are indicated by vertical lines,reduced expression is indicated by horizontal lines, and a combinationof horizontal and vertical lines indicate substantially unchangedexpression. Induction levels of the three p53-regulated genes MDM2,CIP1/WAF1 and BAX were also measured for comparison. CIP1/WAF1 isregulated by both p53-dependent and independent mechanisms, while BAXinduction by IR appears to require p53 plus an “apoptosis-proficiency”factor frequently present in cells that undergo rapid apoptosis afterIR. These three genes were more responsive in the p53 wildtype (wt)lines, with little or no IR-responsiveness in the p53 deficient lines.As reported previously, strong BAX induction was only seen inmyeloid-lymphoid lines with weaker induction in MCF-7 cells. In the caseof the SR leukemia line, constitutive BAX expression was approximatelyan order of magnitude greater than in the other lines, and appreciableinduction was not seen. In contrast to these previously described genes,the ionizing radiation response of the newly characterized genes showsfar more heterogeneity among different cell lines (FIG. 5A). Hence thepresent method can be used to select genes that are expressed in certaincells types (such as myeloid or other blood cells) that will be sampledin subjects who have been or have potentially been exposed to ionizingradiation.

While all of the newly-identified genes respond to IR in at least onecell line in addition to ML-1, two of the cell lines, K562 and A549, didnot show γ-ray regulation of any of the newly-defined IR-responsivegenes, and only ATF3 was induced by any stress agent tested in these twolines (FIG. 5B). Interestingly, K562 was the least responsive of any ofthe cell lines, and was the only line derived from a patient withchronic myelogenous leukemia. Of the nine genes screened, five: SSA T,c-Myc promoter binding protein (MBP-1, or PRDII-BF1), cellular inhibitorof apoptosis 1 (c-IAP1), RELB and BCL3 were primarily induced by IR inthe 12 cell lines examined. Fos-related antigen-1 (FRA-1), RCH1, andprohormone convertase1 (PC1) or neuroendocrine convertase1 (NEC1), allshowed regulation by the base-damaging agents MMS or UV radiation insome cell lines (FIG. 5B). The most wide ranging response was seen foractivating transcription factor 3 (ATF3), which was induced by both MMSand UV radiation in all cell lines tested. With such a strong andpervasive response, ATF3 was selected as playing an important role ingeneralized genotoxic stress responses. Such a strong and pervasiveresponse is a factor that can be used in selecting a probe for thearrays of the present invention.

This example illustrates that a single cell type may provide a lessoptimal model for cellular response to genotoxic stress, such as IR.However, using the techniques in this example, genes can be chosen whichare often or usually induced in response to a genotoxic stress, such asradiation exposure. Such identified genes can be incorporated into aprobe set of a microarray.

In addition, particular cells to be sampled (such as peripheral bloodlymphocytes, see EXAMPLE 9) can be studied to determine the genes thatare differentially expressed in those cells in response to irradiation.Those genes identified as being differentially expressed in response toirradiation can be incorporated into a probe set of a microarray, whichwould be specific for the particular cell type to be tested. Forexample, a microarray can be developed which contains probes which aredifferentially expressed in a specific type of cell (for exampleperipheral blood mononuclear cells such as lymphocytes) in response toirradiation. Such a microarray can then be used to determine/monitorradiation exposure in that cell type.

The large number of probes in the probe set also overcomes the problemof using induction in a single cell line as evidence of radiationexposure induction. Hence a review of the results shown in FIGS. 5A and5B illustrates that MDM2 and CIP1/WAF1 are suitable for incorporationinto a probe set for detecting radiation induced stress in p53 wild typecells, because their expression is substantially induced in all or manyof the cell lines tested. REL-B is also shown to be generallyinformative in both p53 mutants and wild type cells.

EXAMPLE 3 Dose Response Curves

This example describes methods used to generate a dose response curvewhich can be used to determine the time period during which a gene isdifferentially expressed following exposure to ionizing radiation. Suchinformation is useful in determining which probes to incorporate intothe probe set, particularly if the probe set is prepared for analysis ofpotential biological damage at a given time period after a known orsuspected exposure. The genes studied in this example, CIP1/WAF1,GADD45, MDM2, ATF3 and BAX, were chosen since they were shown in theprevious example to be differentially regulated in response toirradiation.

ML-1 cells were grown and exposed to various doses of radiation atapproximately 5.1 cGy/min, to total doses of 2-50 cGy using the MarkI-68 ¹³⁷Cs source as described in EXAMPLE 1. Gene induction was measuredby incubating the irradiated cells at 37° C. for a predetermined numberof hours (such as 1-4, or 24-48 hours following irradiation) followed byRNA extraction using a modified guanidine thiocyanate method (asdetailed in Chomczynski and Sacchi, Anal. Biochem. 162:156-9, 1987).Gene expression was measured by quantitative dot-blot hybridization.Serial dilutions of RNA were immobilized on nylon membranes, hybridizedwith cDNA probes at 55° C. in a buffer containing 50% formamide(Hybrisol I, Oncor, Intergen, Purchase, NY), and washed under standardconditions. Hybridization is quantitated on a phosphorimager (MolecularDynamics, Piscataway, N.J.), and relative signal levels, normalized tothe polyA content of each sample, determined using the RNA-Thinkprogram. The values for relative RNA levels are directly proportional toRNA abundance, and differences of 1.5-fold or more can reliably bemeasured.

Following treatment with 20 Gy γ-rays, the CIP1/WAF1, GADD45, MDM2, ATF3and BAX genes reached maximal induction four hours after irradiation,then declined rapidly until they reached basal levels by 24 hours. Theinduction of CIP1/WAF1 (FIG. 6A) and GADD45 (FIG. 6B) by lower doses ofγ-rays was more rapid than the induction seen with supra-lethal doses,reaching maximum levels two to three hours post-irradiation, and in mostcases beginning to decline after four hours.

Relative induction of expression can be correlated to dosage, as shownin FIGS. 7A, 7B and 8 for several different genes. The maximal inductionof CIP1/WAF1 and GADD45 was approximately linear between 2 and 50 cGy,and showed no indication of a threshold for gene induction (FIG. 7A).Significant induction over basal control levels was observed for bothgenes at all doses. A more gradual dose-dependent response was observedfor the other genes tested (FIG. 7B). MDM2, BAX, and ATF3 were inducedbetween two and three hours following low doses of IR, but 50 cGy onlyresulted in a 2-3 fold increase in the expression of these genes overcontrol levels (FIG. 7B). The lower inducibility of these genes by lowdoses of radiation was consistent with their induction by high doses,which was considerably less than that of CIP1/WAF1 and GADD45 (Table 3).The maximal induction appeared linear between 5 and 50 cGy for MDM2, andbetween 10 and 50 cGy for the other genes. Below these doses, inductionfell off sharply, dropping below the limit of accurate quantitation bythis technique, previously shown to be approximately 1.5-fold.

A broader dose range was next tested for the induction of CIP1/WAF1 todetermine the point at which the induction response begins to saturate.Induction continued to increase in a dose-dependent manner up toapproximately 250 cGy (FIG. 8A). Beyond this point, the dose-dependentincrease in relative induction leveled off sharply, although a trend ofincreased induction appeared to continue up to 20 Gy, the highest dosetested.

The extrapolation of data gathered at high doses to predict effects atlow doses can present difficulties. In particular, it cannot necessarilybe assumed that the dose-response relationship observed at high dosesapplies to the entire dose spectrum. For example, very low doses ofionizing radiation may be more toxic per cGy than higher doses.Therefore, extrapolation from survival at high doses may not predict thelow-dose hypersensitivity revealed by the more accurate methods. Thisresponse may reflect an induction of radio-resistance, perhaps throughinducible DNA repair, which requires a certain threshold dose to betriggered in some cell lines. Understanding the mechanistic basis forsuch induced resistance could have broad implications in areas from riskassessment to cancer treatment.

Low-dose hypersensitivity and induced radio-resistance may be related tothe adaptive response to ionizing radiation, another potentiallyimportant physiological effect of low dose exposures. Exposure to a“priming” or “adapting” dose, usually in the range of 1-25 cGy, reducesthe effects of a subsequent higher “challenge” dose (Stecca and Gerber,Biochem. Pharmacol. 55:941-51, 1998).

The phenomena of low-dose hypersensitivity and radio-adaptation raisethe question of the requirement for a minimum threshold dose to induce atranscriptional response. The threshold effect is a factor to beconsidered for modeling low dose effects from results gathered at highdoses, as the existence of a threshold implies that a dose can beidentified below which exposure carries no risk of response (at leastfor the endpoint under consideration). The experiments showing low dosehypersensitivity have been interpreted to indicate a threshold, usuallyin the neighborhood of 25-30 cGy, which is required to activateinducible repair. Doses below this threshold are proportionately moretoxic than doses which trigger the putative repair system.Radio-adaptive protection against cytogenetic aberrations or cellkilling, however, has been shown to occur following doses as low as 1-2cGy, indicating a much lower or even absent threshold for induction ofthis effect, and perhaps a distinct mechanism of action for the twophenomena.

While the genes studied in this example have not been implicated ininduced repair, changes in mRNA levels in response to radiation in thedose range relevant to these phenomena have been observed. The datareveal no indication of a threshold for the induction of the genesstudied. CIP1/WAF1 and GADD45 were significantly induced by as little as2 cGy with a linear dose-response through 50 cGy (FIG. 7A). For MDM2,ATF3 and BAX, however, the possibility of a threshold for inductioncould not be distinguished from the noise of the assay (FIG. 7B). Anadditional complication to the interpretation of this data is that it iscurrently unknown if a threshold exists for biological significance ofrelative gene expression levels. It is likely that no generalized answerto this question will apply to all genes in all cell lines or celltypes.

Although no evidence of a minimum threshold for induction of CIP1/WAF1was observed, there was an upper limit to the linear increase ininduction, as the induction by 50 cGy was about half that previouslyobserved by 20 Gy (FIG. 7A). In experiments covering a wider range ofdoses from 10 cGy to 20 Gy, a saturation of induction of approximately250 cGy was observed (FIG. 8A). At this dose, less than 5% of the cellsretain mitotic viability. The coincidence of saturation for geneinduction with nearly complete cell killing raises the issue of whethersurviving cells contribute to the transcriptional response followingeven low doses of ionizing radiation, or if gene induction occurs onlyin lethally damaged cells. To address this question, the relativeinduction per lethally damaged cell which would be required to producethe inductions observed in the treated population as a whole, if themeasured gene induction were due entirely to the response of lethallydamaged, reproductively dead cells was calculated (FIG. 8B). For thedoses where surviving fractions could not be measured directly, theywere predicted from the survival curve (FIG. 1). In this model, eachcell responding to 2 cGy would have to produce a relative induction ofCIP1/WAF1 greater than 100-fold over the basal level to result in theobserved induction of approximately 3.5-fold across the wholepopulation. This relative induction per responding cell would then haveto decrease with increasing dose through 50 cGy. Furthermore, as theproportion of the population suffering lethal damage increased further,the induction per responding cell would level out, then begin toincrease again at doses above 5 Gy. A similar relationship was alsocalculated for the dose-response of GADD45 induction (FIG. 8B). If themeasured gene induction were due entirely to the response of lethallydamaged cells, the relative induction per lethal event would be expectedto remain constant with changing dose and surviving fraction, so therelative induction of cell killing in an irradiated population would notappear to account for the observed increases in mRNA levels.

The contribution of cells which survive the treatment to the geneinductions measured at low doses would also be consistent with thedisturbances in cell cycle progression observed at all doses tested.Even with 2 cGy, there was a transient decrease of S-phase cells in thepopulation. This decrease corresponded to a larger proportion of thepopulation than the predicted non-surviving fraction. A transient G1delay following a consistent trend in both dose-response and temporalkinetics was observed (FIG. 3B). While the lowest dose used, 2 cGy,resulted in a brief accumulation of cells in G1, accumulation of cellsin G2 appeared to require a higher dose. CIP1/WAF1, the transcript moststrongly induced by low doses of γ-rays, is a major mediator of G1arrest (Di Leonardo et al. Genes Dev. 8:2540-51, 1994; Waldman et al.Cancer Res. 55:5187-90, 1995). Thus, the observed G1 delay is consistentwith this CIP1/WAF1 induction. A complete understanding of low-doseeffects should not come solely from high dose experiments, but insteadmay require further careful studies in the low dose range.

Measurable differential gene expression effects were observed atrelatively low, biologically relevant doses, such as 2 cGy. Inradiotherapy the standard daily dose is about 2 Gy. Therefore, theresults of these experiments indicate that the expression of many genesis altered at this dose. The probe sets of the present invention iscapable of measuring exposure to relatively low doses of ionizingradiation (for example less than 2 Gy or 1 Gy) that may be considerednon-toxic because of the absence of acute effects, but which may haveshort term effects that induce differential gene expression, and mayhave long term effects (such as carcinogenesis).

This example illustrates that probe sets that measure not only the factof exposure to radiation, but also a probable dose of exposure toionizing radiation, can be created by identifying genes that aredifferentially expressed at certain radiation doses. For example, probesets can be designed which contain genes which are only differentiallyexpressed at higher doses of irradiation (for example 20 Gy), or geneswhich are differentially expressed at lower doses of irradiation (forexample 1-50 cGy). Furthermore, the methods for generating dose responsecurves described in this example can be used to select probes for theprobe set, based on the probable time after radiation exposure occurred.Curves that show sustained or maximal expression 12, 24 or 48 hoursafter irradiation would identify probes that are useful for evaluationof genotoxic stress from exposure to ionizing radiation at times whenclinical evaluations are likely to occur. In addition, patterns of probeset hybridization can also be associated with specified times followingprobable irradiation. After identifying the genes of interest, they canbe incorporated into a probe set of a microarray.

EXAMPLE 4 p53-Associated Components of IR-Induction

This example demonstrates how to determine if the IR-induction of FRA-1and ATF3 involve a p53 regulatory component. FRA-1 was induced by IR insome of the wt p53 lines, but was not induced in any of the p53 mutantlines studied (FIGS. 5A and 5B). In addition, the 5-fold IR-induction ofATF3 in RKO was attenuated in the RKO/E6 cell line, although someIR-induction was observed among the other p53 mutant cell lines. FIG. 9Ashows the IR-induction of ATF3 in the p53 wild-type human carcinoma cellline RKO and in RKO/E6, in which p53 function has been abrogated by anE6 expression vector. RKO/E6 lacks appreciable functional p53. Thedisruption by E6 of ATF3 induction by IR supports a role for p53 in itsinduction.

To further examine the extent of dependence of ATF3 IR-induction on p53status, in vivo induction was examined in wild-type and p53−/−(knockout) mice (Donehower et al., Nature 356:215-21, 1992) using 5 Gywhole-body γ-irradiation. While ATF3 was well induced by two hours afterirradiation in the thymus of wild-type mice, there was no significantinduction in the p53−/− mouse (FIG. 9A). ATF3 levels remained elevatedat four and eight hours after irradiation in the thymus of wild-typemice, without any induction in the p53−/− mice. A similar trend wasobserved in the liver, where ATF3 was induced about 4-fold in thewild-type mouse, but showed no induction in the p53 knockout.

ATF3 is a member of the activating transcription factor/cAMP responseelement binding protein (ATF/CREB) family which homodimerizes to represstranscription from promoters with ATF sites. An alternatively splicedform of the ATF3 transcript, which lacks DNA binding activity, is alsoexpressed in cells, but this form promotes transcription (Chen et al.,J. Biol. Chem. 269:15819-26, 1994). The sizes of ATF3 transcriptshybridizing on the northern blot demonstrated that the smalleralternatively spliced form was the major transcript expressed inuntreated ML-1 cells, whereas the IR-induced transcript waspredominantly of the full length form.

The induction of ATF3 by the DNA-damaging agents MMS and UV radiation inall 12 cell lines examined (FIG. 5) extends the range of stressresponses in which ATF3 is involved. Its wide-ranging responsiveness issimilar to CIP1/WAF1 and GADD45, which are stress inducible by bothp53-dependent and independent mechanisms, and highlights the complexityof ATF3 regulation.

Although FRA-1 was not IR-inducible in RKO cells, a similar comparisonwas possible using the MCF7 and MCF7/E6 cell lines. The human breastcarcinoma cell line MCF7 has wild-type p53, while MCF7/E6 has lackedappreciable wild-type p53 function. The reduced IR-induction of FRA-1 inMCF7/E6 compared to MCF7 supports a role for p53 in the IR-induction ofthis gene (FIG. 9B). Furthermore, whole-body γ-irradiation of wild-typemice resulted in FRA-1 induction in the thymus, but no inductionoccurred in the thymus of the p53−/− (FIG. 9B). Such results can be usedto select certain probes (for example FRA-1) for detection of whole-bodyradiation. FRA-1 is an immediate early gene induced by serumstimulation, the product of which shares several regions of amino acidhomology with Fos. It is also down-regulated by UVB and upregulated byUVA. Because of its homology to Fos, and the involvement of p53 in itsIR-induction, FRA-1 the use of the present assay can be taken todemonstrate a link between p53 and AP1 function and the MAPK pathway.

MBP-1 represented another potentially p53 regulated gene, showing apattern similar to that seen for BAX or BCL-X, in that it was inducedonly in p53-wild-type cell lines of lymphoid or myeloid lineage. Theinduction of the murine homolog of MBP-1 in the tissues of wild-type andp53−/− mice was observed to have marginal to absent expression in liverand thymus, but strong expression in spleen. Treatment with ionizingradiation resulted in a 2-fold induction of this gene in the spleens ofboth p53 wild-type and p53−/− mice, suggesting that this gene does notrequire p53 function for its induction, but that its expression andinduction are both limited to a subset of cell types. This would beconsistent with a role for MBP-1 in tissue specific p53-independentstress responses.

The finding that only two of nine of these genes examined in this cellline panel showed a recognizable p53 component to their regulationbelies the recent focus of stress-gene studies primarily onp53-regulated genes. In light of the loss of functional p53 in themajority of tumors, the non-p53-dependent stress response genes appearsto also be an important consideration in cancer treatment.

An advantage o the present invention is its ability to examine bothp53-regulated and non-p53 regulated genes, or other combinations of genetypes (for example genes which are recognized oncogenes or genes whichare not recognized oncogenes). Quantitative functional genomicsapproaches, such as cDNA microarray hybridization, can also be used incombination with the radiation sensor array to unravel theinter-relationships of the molecular response pathways involved.Although radioactive-probe hybridization to nylon filter arrays providesa useful method to screen for potential genes of interest which differin expression levels between two samples, differential screening has itsown limitations (Fargnoli et al., Anal Biochem 187:364-73, 1990) some ofwhich are avoided by the use of probes labeled with differentfluorochromes co-hybridized to the same microarray. Other methods foridentification of differentially expressed mRNAs, such as differentialdisplay, subtractive library hybridization and serial analysis of geneexpression (SAGE) (Velculescu et al., Science 270:484-7, 1995), can bebiased toward detection of highly-expressed and/or strongly-inducedtranscripts.

With the microarray approach of the present invention, quantitativeresults over a wide dynamic range were obtained for many genes. Theapplication and further refinement of quantitative fluorescent cDNAmicroarray hybridization have the potential to advance our understandingof the fields of stress gene response and radiation biology, and toextend this technology beyond simple pair-wise comparisons toapplications such as tumor typing, pharmacological screening,biomonitoring, and rapid carcinogen screening.

This example combined with the results from EXAMPLE 2 illustrate thatprobe sets that measure differential expression in response to abiologically significant amount of radiation for specific tumors can begenerated. Such probe sets can be used to monitor a subject who isundergoing radiotherapy for the treatment of a tumor. For example, probesets can be designed which contain genes from a specific type of tumor,which are differentially expressed in response to irradiation of thetumor cells. This probe set would then be used to monitor a subject'sresponse to the radiotherapy. Further details are provided in EXAMPLE10. As an extension of this probe set, the present method can be used in“tumor profiling,” wherein gene expression profiles are used to predictthe most effective treatment for each individual subject. In thismethod, cells would be isolated from the tumor in a subject, and thedifferential expression of the genes in that tumor in response toirradiation measured as described in the examples herein. A pattern ofdifferential expression found to be associated with a particulartherapeutic response to radiation therapy can then be used as a factorwhen considering whether to include radiation therapy in the treatmentof the tumor. For example, a pattern of differential expressionassociated with a good response to radiation therapy could be used toindicate that radiation therapy should be instituted.

EXAMPLE 5 New Radiation Responsive Sequences

Using the ML-1 cancer cell line and the cDNA microarray hybridizationtechnology described in this specification, 30 sequences from ML-1 wereidentified that were not previously known to be radiation-responsive.These sequences are Image ID Nos. 428248, 129632, 52681, 52530, 110503,236422, 153355, 52753, 51363, 161023, 35326, 487130, 291503, 41452,23464, 243143, 205633, 21420, 48677, 43060, 233194, 295093, 48085,26541, 28089, 359119, 28116, 80549, 485963, 24927. Arrays can be madethat incorporate these sequences, or probe fragments thereof, along withcontrols and/or other sequences that are already known to be informativeabout radiation exposure, and/or dosage of radiation exposure.

EXAMPLE 6 Late Induced Genes

This example describes detection of differential gene expression inirradiated ML-1 cells using different microarrays. Although the specificexample discloses expression in ML-1 cells, this method can be used todetect differential gene expression in any cell type.

ML-1 cells were grown and irradiated as described in EXAMPLE 1. LabeledcDNA subsequently prepared from the irradiated cells was exposed tovarious arrays as described in EXAMPLE 2. cDNA microarray hybridizationstudies were performed with the 1.2K chip described in EXAMPLE 2 whichcontained 1,200 probes in a probe set (Table 1), a 5K chip whichcontained 5,000 probes in a probe set, and a 7K chip which contained7,000 probes in a probe set. The 5K and 7K chips containedrepresentative verified expressed ESTs from the human genome, and weredesigned to permit as much as possible of the human genome to bescreened using irradiated ML-1 cells. The arrays used represent thelargest sequence verified clone sets available to print at the time ofthe experiments. As more clones become available, they are added to newprints to be screened. The 1.2K array was a smaller set of ESTs designedfor the same purpose (see EXAMPLE 2).

Table 4 shows the clones used to make the 1.2K array along with datashowing the relative expression detected at each position of the array.The clones used to make the 5K array are identified in Table 5 and forthe 7K array in Table 6. Although these particular arrays were used inthese experiments, other arrays may be used. For example, as additionalarrays become available that include even more ESTs, additionalbiomarkers of radiation exposure can be found and added to the probesets disclosed in Tables 9-13. Moreover, the 1.2K, 5K, and 7K arrays canalso be used with different cells (such as human peripheral lymphocytes)to screen for other markers that would be even more informative whentesting those cells. TABLE 4 Results from the 1.2K microarray. Image IDor array clone name ratio* Waf1 42.02 268652 18.76 428248 14.53 42824811.32 151767 9.48 129632 9.29 52681 7.17 512391 6.92 52530 6.37 1105034.83 328692 4.82 124554 4.73 236422 4.64 153355 4.32 26474 4.20 3094774.17 52681 4.03 52753 3.40 280376 3.39 51363 3.38 470602 3.23 1470753.17 161023 3.13 35326 3.12 HIV F2 3.10 470062 3.03 120466 3.02 CoT13.01 486074 2.93 112500 2.83 471252 2.81 487130 2.77 291503 2.76 Mdm22.72 41452 2.65 52681 2.64 Bak 2.57 23464 2.53 243143 2.51 205633 2.51284031 2.49 21420 2.47 48677 2.41 485963 2.34 162772 2.32 Hu placental2.32 51439 2.30 126379 2.29 253545 2.27 80549 2.26 Bcl-xl 2.26 1561032.25 489395 2.24 24927 2.23 27899 2.19 427923 2.17 286249 2.15 531642.14 470685 2.10 47679 2.09 116906 2.08 HIV F8 2.07 21531 2.04 2807682.04 178508 2.03 512472 2.03 121849 2.02 42906 2.01 52650 2.01 1455032.01 110022 1.98 343311 1.98 149910 1.98 137531 1.97 mouse c-kit 1.97231292 1.94 mouseEDNRB 1.94 290871 1.92 52193 1.91 26871 1.91 1528441.91 23173 1.90 49164 1.89 512385 1.89 289606 1.89 108837 1.88 2831161.86 361381 1.86 51746 1.85 187987 1.85 132420 1.84 299198 1.84 1544201.84 33941 1.83 376218 1.83 140358 1.83 116781 1.83 154420 1.83 3597691.82 471494 1.81 486844 1.81 486716 1.80 308392 1.79 empty 1.79 HIV F41.79 47229 1.78 39920 1.78 486785 1.77 203132 1.77 200209 1.76 1235861.76 60298 1.76 296642 1.75 146307 1.74 429539 1.74 260288 1.73 735151.72 53173 1.72 HIV F12 1.72 322604 1.71 125187 1.71 417357 1.71 775771.70 279954 1.70 417403 1.70 428231 1.69 120157 1.69 321242 1.68 2645761.68 486199 1.68 158970 1.68 153025 1.68 41959 1.67 72869 1.67 empty1.67 HIV F5 1.66 53184 1.66 327111 1.66 307342 1.66 155717 1.66 Alk 1.65135118 1.65 183661 1.65 34974 1.65 52162 1.64 22790 1.64 mouse c-met1.64 154472 1.64 22036 1.63 35516 1.63 362702 1.63 417592 1.63 464271.63 360531 1.63 359793 1.63 298371 1.61 486161 1.61 310141 1.61 225891.61 418111 1.61 139073 1.61 31687 1.61 289428 1.61 366539 1.60 5122761.60 127821 1.60 mouse EDN3 1.59 377275 1.59 109957 1.59 147744 1.59258589 1.58 29627 1.58 53358 1.58 306134 1.58 295389 1.58 21656 1.57127089 1.57 30966 1.56 mouse pme117 1.56 470730 1.55 471822 1.55 432311.55 HIV F9 1.54 35665 1.54 154015 1.54 158970 1.54 363805 1.53 366761.53 47306 1.53 28774 1.53 209655 1.53 488103 1.53 50603 1.53 4695431.53 143751 1.52 284546 1.52 51825 1.51 22012 1.51 509731 1.51 1446931.51 138917 1.51 126542 1.51 38573 1.50 36927 1.50 236420 1.50 4888011.50 137575 1.50 86017 1.49 31061 1.49 303109 1.48 22589 1.48 35865 1.48359466 1.48 238577 1.48 302490 1.48 40442 1.48 36593 1.47 344482 1.47471715 1.47 288650 1.47 470251 1.47 364110 1.46 154763 1.46 36809 1.4636904 1.45 37797 1.45 298371 1.44 253869 1.44 21468 1.44 50503 1.44270980 1.44 345928 1.44 35559 1.44 mouse pdgfra 1.43 366420 1.43 2059051.43 37347 1.43 288797 1.43 50941 1.43 37513 1.42 33415 1.42 221619 1.41429976 1.41 136769 1.40 44180 1.40 254187 1.40 362009 1.39 30981 1.39272110 1.39 51332 1.39 49725 1.39 472067 1.38 49967 1.38 36074 1.3834396 1.38 418138 1.38 49509 1.38 154441 1.38 357807 1.38 281029 1.37syn cDNA 33 1.37 48803 1.37 46171 1.37 53155 1.36 429142 1.36 26811 1.36syn cDNA 33 1.36 32427 1.35 47874 1.35 270148 1.35 489419 1.35 2664861.34 48398 1.34 34247 1.34 31855 1.34 lambda 1.34 162479 1.34 41439 1.34193901 1.34 376303 1.34 254428 1.34 299539 1.34 510381 1.34 25880 1.33364752 1.33 178169 1.33 25495 1.33 510258 1.33 39285 1.33 204301 1.3326992 1.33 268412 1.33 26801 1.33 358168 1.33 44495 1.33 40117 1.33182633 1.32 mouse ece 1 1.32 264554 1.32 31210 1.32 49576 1.32 3009731.32 44307 1.32 288733 1.32 323001 1.31 22379 1.31 487264 1.31 3463961.31 44193 1.31 51293 1.31 82991 1.30 34217 1.30 27944 1.30 40562 1.30363539 1.30 50503 1.30 264200 1.30 50816 1.30 417689 1.30 258790 1.30417218 1.30 124927 1.30 lambda 1.29 35284 1.29 203017 1.29 213597 1.2938393 1.29 509882 1.29 154536 1.29 128100 1.29 296021 1.28 52587 1.2835609 1.28 359412 1.28 159833 1.28 33368 1.27 376781 1.27 512294 1.27234198 1.27 empty 1.27 49666 1.27 162584 1.26 365147 1.26 268727 1.26183950 1.26 489042 1.26 44722 1.25 486510 1.25 179711 1.25 41208 1.25320903 1.25 23866 1.25 53024 1.25 47359 1.24 191007 1.24 48097 1.24344109 1.24 198205 1.24 46019 1.24 27624 1.24 HIV F11 1.24 236338 1.2432304 1.23 322452 1.23 syn cDNA 10 1.23 115277 1.23 201673 1.23 mouse1.23 aim 1/2.2 46354 1.23 428404 1.23 471174 1.23 43229 1.22 306170 1.2239148 1.22 249348 1.22 51686 1.22 253773 1.21 322000 1.21 25485 1.21361692 1.21 47343 1.21 510130 1.21 133122 1.21 37435 1.21 340947 1.21syn cDNA 31 1.21 209310 1.21 234357 1.21 298314 1.21 302394 1.21 3602011.20 42927 1.20 47499 1.20 39086 1.20 487097 1.20 219735 1.20 4885961.20 37962 1.20 49888 1.19 31988 1.19 51687 1.19 34761 1.19 34860 1.19488989 1.19 377441 1.19 108284 1.19 36844 1.19 510467 1.19 290117 1.1923185 1.19 241412 1.19 290724 1.19 40026 1.19 42363 1.19 356890 1.1925081 1.18 mouse 1.18 tyrosinase HIV F13 1.18 323162 1.18 363799 1.18193736 1.18 48077 1.18 323555 1.18 37508 1.18 50666 1.18 44477 1.18429926 1.18 45794 1.17 CoT1 1.17 35110 1.17 509820 1.17 46042 1.17509682 1.17 512244 1.17 42648 1.17 40304 1.17 359077 1.17 469731 1.17364975 1.16 82850 1.16 488373 1.16 110589 1.16 37231 1.16 52629 1.16231497 1.16 286197 1.16 144944 1.16 298314 1.16 48452 1.16 510101 1.16342256 1.16 358467 1.16 305455 1.16 40831 1.15 47493 1.15 375922 1.15252185 1.15 39542 1.15 37679 1.15 33176 1.15 36291 1.15 469256 1.15mouse slug 1.15 27848 1.15 195889 1.15 80549 1.14 42910 1.14 365647 1.1432531 1.14 247869 1.14 52019 1.14 324700 1.14 26585 1.14 262691 1.1432774 1.14 33267 1.14 51318 1.14 36560 1.14 125555 1.14 37904 1.14 323191.13 36922 1.13 51666 1.13 260052 1.13 361692 1.13 36975 1.13 1804471.13 30272 1.13 23692 1.13 44178 1.13 42576 1.13 36007 1.13 empty 1.1344266 1.13 syn cDNA 10 1.13 49318 1.13 34005 1.12 40670 1.12 324815 1.12272110 1.12 427750 1.12 125676 1.12 49584 1.12 359171 1.12 470493 1.12361728 1.12 42532 1.12 44710 1.11 51528 1.11 307471 1.11 mouse mart11.11 241489 1.11 242134 1.11 487965 1.11 43858 1.11 259241 1.11 4710961.11 49777 1.10 231292 1.10 43129 1.10 23731 1.10 53084 1.10 53107 1.10271416 1.10 syn cDNA 10 1.10 44218 1.10 empty 1.10 429368 1.10 3642781.09 22260 1.09 26052 1.09 84730 1.09 25517 1.09 50032 1.09 52489 1.0930592 1.09 50117 1.09 135381 1.09 44105 1.09 427686 1.09 346587 1.0924110 1.09 470185 1.09 49665 1.09 33477 1.09 40765 1.08 29204 1.08 474751.08 40493 1.08 79742 1.08 39195 1.08 271416 1.08 30885 1.08 43711 1.0823073 1.08 52773 1.08 syn cDNA 8 1.08 39796 1.08 36232 1.08 44367 1.08231302 1.08 40205 1.08 44414 1.08 27715 1.08 33643 1.08 26599 1.08347751 1.08 26599 1.08 33525 1.08 29054 1.07 31116 1.07 36950 1.07320514 1.07 45564 1.07 488658 1.07 26593 1.07 488904 1.07 30730 1.07 syncDNA 30 1.07 418436 1.07 51373 1.07 syn cDNA 31 1.07 470470 1.06 367001.06 38966 1.06 syn cDNA 30 1.06 syn cDNA 33 1.06 51961 1.06 34637 1.0630622 1.06 39380 1.06 282065 1.06 46329 1.06 486410 1.06 syn cDNA 381.06 40008 1.06 37721 1.06 47110 1.06 37892 1.06 33831 1.06 51940 1.0541094 1.05 114116 1.05 271478 1.05 232714 1.05 43719 1.05 343871 1.0526659 1.05 24652 1.05 306013 1.05 42677 1.05 47202 1.05 31852 1.04362926 1.04 39173 1.04 52222 1.04 22545 1.04 26789 1.04 258747 1.0453068 1.04 418105 1.04 30852 1.04 35850 1.04 33632 1.04 53238 1.04364982 1.03 486086 1.03 43338 1.03 HIV F6 1.03 HIV F10 1.03 45840 1.0323804 1.03 52896 1.03 417136 1.03 empty 1.03 40111 1.03 29629 1.02 346471.02 41565 1.02 23831 1.02 204681 1.02 44014 1.02 145932 1.02 32875 1.0226503 1.02 357374 1.02 429057 1.02 32198 1.01 22040 1.01 36681 1.0147333 1.01 50271 1.01 375843 1.01 376725 1.01 42059 1.01 39861 1.0145941 1.01 33274 1.01 271198 1.01 24886 1.01 33690 1.01 33438 1.01 246091.01 470817 1.01 49691 1.01 110589 1.00 310428 1.00 25400 1.00 syn cDNA8 1.00 52338 1.00 32717 1.00 323500 1.00 27473 1.00 295208 1.00 syn cDNA25 1.00 lambda 1.00 32822 1.00 46303 1.00 31242 1.00 42352 1.00 1943841.00 36844 0.99 44537 0.99 347573 0.99 485690 0.99 486335 0.99 407640.99 30573 0.99 190711 0.99 50576 0.99 26087 0.99 41773 0.99 syn cDNA 310.99 43549 0.99 50363 0.99 52489 0.99 39827 0.99 427943 0.99 53128 0.9942130 0.99 52931 0.99 46302 0.99 pUC18 0.99 50609 0.99 27681 0.99 359330.99 empty 0.99 53185 0.99 301723 0.99 31155 0.99 33803 0.99 empty 0.9938829 0.98 34005 0.98 36916 0.98 44563 0.98 42993 0.98 syn cDNA 31 0.9836790 0.98 366576 0.98 418320 0.98 52342 0.98 487878 0.98 37196 0.9839781 0.98 341763 0.98 37796 0.98 470635 0.98 31454 0.98 51432 0.9836109 0.98 46777 0.98 syn cDNA 38 0.98 305455 0.98 47384 0.97 3000510.97 85906 0.97 29640 0.97 Bcl-2 0.97 377181 0.97 aim2/IFI16 0.97 2093100.97 380738 0.97 415731 0.97 345600 0.97 40151 0.97 26167 0.97 283090.97 33692 0.97 429861 0.97 126519 0.97 357909 0.97 33620 0.97 251540.96 51785 0.96 51814 0.96 484993 0.96 25584 0.96 486560 0.96 50322 0.96415679 0.96 343166 0.96 32898 0.96 471631 0.96 33183 0.96 31116 0.9625169 0.95 366981 0.95 38567 0.95 44605 0.95 44666 0.95 239287 0.9537234 0.95 45421 0.95 327396 0.95 35623 0.95 44159 0.95 48713 0.95427943 0.95 35297 0.95 23332 0.95 37451 0.95 31131 0.95 42058 0.95259291 0.95 46574 0.95 51241 0.95 486233 0.94 196543 0.94 49898 0.9443601 0.94 200378 0.94 syn cDNA 25 0.94 323648 0.94 48717 0.94 463390.94 30691 0.94 328467 0.94 39965 0.94 427857 0.94 Gadd153 0.94 514180.94 syn cDNA 25 0.94 361401 0.93 38876 0.93 49788 0.93 32609 0.93 326260.93 33008 0.93 39686 0.93 50555 0.93 23346 0.93 30802 0.93 47038 0.9344302 0.93 syn cDNA 30 0.93 36202 0.93 34327 0.92 267246 0.92 34141 0.92307741 0.92 301363 0.92 26503 0.92 32649 0.92 37230 0.92 45222 0.92324698 0.92 37269 0.92 376737 0.92 29799 0.92 488891 0.92 41392 0.9241813 0.92 23019 0.92 140827 0.92 37433 0.92 HIV F7 0.92 46213 0.9236393 0.92 syn cDNA 8 0.92 44311 0.92 42888 0.92 53316 0.92 486375 0.9149926 0.91 49950 0.91 40959 0.91 357357 0.91 syn cDNA 38 0.91 3237760.91 30588 0.91 35313 0.91 41857 0.91 53322 0.91 47125 0.91 357442 0.9140440 0.91 38805 0.90 49897 0.90 51540 0.90 TRP1 0.90 syn cDNA 10 0.9028985 0.90 26599 0.90 22293 0.90 26147 0.90 41199 0.90 415636 0.90 306640.90 46419 0.90 485192 0.90 39903 0.90 42615 0.90 512429 0.90 3470320.90 359465 0.90 302157 0.90 empty 0.90 43110 0.89 361815 0.89 505760.89 40991 0.89 44699 0.89 321246 0.89 34537 0.89 43409 0.89 325182 0.8936215 0.89 syn cDNA 30 0.89 52415 0.89 mouse 0.89 mi/microphtha lmia33045 0.89 37380 0.89 42096 0.89 471889 0.89 322029 0.89 42214 0.8926922 0.89 35483 0.89 37485 0.89 138998 0.89 31238 0.89 40276 0.89 384710.89 27815 0.89 46711 0.89 49322 0.89 51461 0.89 53292 0.89 469272 0.8933943 0.89 44527 0.88 34357 0.88 43129 0.88 32141 0.88 375724 0.88 305780.88 26572 0.88 syn cDNA 8 0.88 43504 0.88 323028 0.88 340644 0.88512458 0.88 39728 0.88 41918 0.88 42284 0.88 37477 0.88 empty 0.88 248990.88 EDNRB 0.88 121147 0.87 324655 0.87 34945 0.87 39270 0.87 3765070.87 39159 0.87 222502 0.87 278409 0.87 343465 0.87 293934 0.87 438260.87 23132 0.87 49161 0.87 510405 0.87 syn cDNA 33 0.87 340878 0.87152762 0.87 296476 0.87 51854 0.86 endothelin 1 0.86 32409 0.86 syn cDNA25 0.86 43021 0.86 51685 0.86 489194 0.86 321308 0.86 32644 0.86 4877770.86 380403 0.86 39169 0.86 35615 0.86 tyrosinase 0.86 43885 0.86 327900.86 27794 0.86 24392 0.86 31154 0.86 37964 0.86 44721 0.85 199381 0.8538859 0.85 21652 0.85 49751 0.85 38647 0.85 486012 0.85 47631 0.85 346710.85 26082 0.85 22611 0.85 114648 0.85 50359 0.85 syn cDNA 38 0.85 420880.85 343646 0.85 37622 0.85 43960 0.85 31143 0.85 42349 0.85 24145 0.85293032 0.85 40450 0.85 51454 0.85 49383 0.85 36717 0.84 26483 0.84213502 0.84 37040 0.84 30012 0.84 30619 0.84 43504 0.84 203184 0.8438500 0.84 27818 0.84 42844 0.84 31863 0.84 81221 0.83 22230 0.83 502340.83 376362 0.83 43241 0.83 256323 0.83 323390 0.83 50203 0.83 230140.83 293274 0.83 44173 0.83 230976 0.83 50888 0.83 340734 0.83 HIV F30.83 279727 0.83 42020 0.83 357435 0.83 33794 0.83 470621 0.83 257250.82 267682 0.82 empty 0.82 489217 0.82 mouse TRP2 0.82 mouse 0.82aim2/IFI16 mouse TRP1 0.82 34606 0.82 487386 0.82 30149 0.82 pUC18 0.82Gadd34 0.82 48458 0.82 291057 0.82 CoT1 0.82 487982 0.82 42380 0.8251302 0.82 Hu placental 0.82 pUC18 0.82 empty 0.82 empty 0.82 51740 0.8132790 0.81 310390 0.81 21810 0.81 49849 0.81 30781 0.81 pUC18 0.81 305430.81 279482 0.81 52652 0.81 223176 0.81 345645 0.81 341130 0.81 509990.81 48503 0.81 280735 0.81 488092 0.81 38397 0.81 47559 0.81 3588480.81 37760 0.81 36060 0.81 51219 0.81 470196 0.81 300960 0.81 3104060.80 25764 0.80 344769 0.80 38840 0.80 47386 0.80 40874 0.80 28573 0.80309615 0.80 362332 0.80 50232 0.80 39205 0.80 43622 0.80 470792 0.8047838 0.80 26366 0.80 30970 0.80 510151 0.80 280465 0.80 50576 0.80509588 0.80 34349 0.79 113941 0.79 471217 0.79 36775 0.79 28776 0.7936796 0.79 38770 0.79 22445 0.79 45851 0.79 47648 0.79 41796 0.79 262860.79 45568 0.79 375724 0.79 49243 0.79 417322 0.79 291736 0.79 314560.79 42894 0.78 29438 0.78 32407 0.78 28595 0.78 45376 0.78 271662 0.7847727 0.78 50171 0.78 ME491 0.78 39884 0.78 340922 0.78 24269 0.78307293 0.78 49608 0.78 40360 0.78 284669 0.78 31164 0.78 43194 0.77297589 0.77 80239 0.77 45643 0.77 281778 0.77 192694 0.77 43541 0.7741356 0.77 123953 0.77 48629 0.77 290493 0.77 28245 0.77 46548 0.7744255 0.77 21477 0.76 299626 0.76 43563 0.76 47833 0.76 112035 0.7632682 0.76 489327 0.76 221690 0.76 257458 0.76 39876 0.76 39993 0.7642295 0.76 27052 0.76 53071 0.76 248258 0.76 359051 0.76 24638 0.7636191 0.76 34255 0.75 26128 0.75 26184 0.75 509710 0.75 27549 0.75116713 0.75 44050 0.75 357239 0.75 31355 0.75 52564 0.74 345527 0.7426129 0.74 43631 0.74 323181 0.74 32684 0.74 415060 0.74 32615 0.7439391 0.74 45604 0.74 510245 0.74 27230 0.74 322000 0.74 470610 0.7427927 0.74 46920 0.74 46936 0.74 44584 0.73 471620 0.73 129268 0.7349980 0.73 261518 0.73 262575 0.73 375635 0.73 26531 0.73 366904 0.7350561 0.73 346305 0.73 28410 0.73 24588 0.73 29234 0.72 366942 0.7250038 0.72 375620 0.72 43092 0.72 45658 0.72 22798 0.72 41559 0.72231089 0.72 32707 0.72 415102 0.72 32750 0.72 416184 0.72 284459 0.7249403 0.72 50308 0.71 366647 0.71 376649 0.71 376110 0.71 Hu placental0.71 37366 0.71 35320 0.71 417124 0.71 31169 0.71 304947 0.71 24642 0.7134660 0.70 48406 0.70 192694 0.70 27511 0.70 48762 0.70 357309 0.70429135 0.70 42700 0.70 53227 0.70 40110 0.70 51504 0.70 34011 0.69 329830.69 257766 0.69 34662 0.69 509710 0.69 244355 0.69 193913 0.68 3104900.68 347685 0.68 471918 0.68 375635 0.68 358538 0.68 322148 0.68 369830.68 41511 0.68 39093 0.68 138604 0.68 51018 0.68 28573 0.67 32658 0.67pdgfr 0.67 201797 0.67 50106 0.67 29435 0.66 40886 0.66 CoT1 0.66 2646060.66 22285 0.66 45291 0.66 190107 0.66 49255 0.66 38983 0.65 298187 0.65376294 0.65 48234 0.65 381219 0.65 471218 0.65 417127 0.64 47884 0.64300071 0.64 356964 0.64 470691 0.63 51621 0.63 45233 0.63 131828 0.63320538 0.63 46449 0.63 HGF 0.63 243159 0.63 45188 0.62 37118 0.62 3647160.61 62186 0.61 40911 0.61 366824 0.61 115336 0.61 138460 0.61 1240860.61 291943 0.61 62186 0.60 47463 0.60 366509 0.60 Hu placental 0.6049277 0.60 34555 0.59 37145 0.59 41792 0.59 43550 0.59 510595 0.59 512090.59 44689 0.58 358677 0.58 34304 0.58 22493 0.58 115408 0.58 4300270.58 51555 0.57 34355 0.57 328330 0.56 248613 0.56 469724 0.56 lambda0.56 32636 0.55 32438 0.54 376285 0.54 22568 0.54 345722 0.54 3099240.54 43060 0.53 415206 0.53 42777 0.53 HIV F1 0.51 487417 0.51 3465340.47 233194 0.46 295093 0.45 48085 0.44 26541 0.43 empty 0.41 28089 0.38248032 0.34 359119 0.27 28116 0.23 empty 0.19 empty 0.18 empty 0.18empty 0.15 417226 0.14 51699 0.12*relative expression levels

TABLE 5 Image ID Numbers Present in the 5K microrray. 20115; 21652;21738; 22012; 22040; 22074; 22260; 22293; 22411; 22493; 22711; 22731;22918; 23019; 23073; 23132; 23173; 23185; 23282; 23431; 23772; 23776;23804; 23831; 23878; 23932; 24004; 24032; 24085; 24145; 24415; 24642;24884; 25499; 25584; 25588; 25679; 25725; 25755; 26021; 26162; 26184;26314; 26366; 26418; 26566; 26566; 26578; 26616; 26617; 26711; 26811;26910; 26922; 27104; 27548; 27549; 27624; 27787; 27848; 28012; 28098;28218; 28309; 28410; 28422; 28469; 28475; 28823; 28985; 29054; 29063;30093; 30170; 30272; 30502; 30664; 30885; 31072; 31093; 31143; 31169;31210; 31251; 31842; 31866; 31873; 32231; 32472; 32609; 32684; 32875;32898; 32996; 33045; 33049; 33051; 33051; 33182; 33299; 33327; 33478;33525; 33632; 33690; 33826; 33941; 33949; 34106; 34255; 34302; 34315;34355; 34357; 34396; 34439; 34616; 34671; 34689; 34773; 34778; 34795;34849; 34852; 34888; 34945; 35077; 35105; 35185; 35191; 35191; 35236;35271; 35483; 35828; 36374; 36387; 36393; 36493; 36607; 36775; 36950;37196; 37234; 37366; 37449; 37451; 37491; 37904; 38465; 38471; 38763;38763; 39093; 39127; 39159; 39173; 39274; 39285; 39593; 39796; 39798;39808; 39884; 39884; 39993; 40017; 40026; 40042; 40056; 40299; 40304;40360; 40365; 40562; 40567; 40580; 40643; 40699; 40704; 40721; 40751;40751; 40781; 40844; 40887; 40946; 41199; 41356; 41452; 41511; 41541;41565; 41591; 41650; 41658; 41672; 41898; 41929; 42059; 42076; 42076;42096; 42118; 42214; 42258; 42313; 42313; 42352; 42373; 42558; 42576;42706; 42739; 42880; 42993; 43021; 43129; 43198; 43207; 43231; 43241;43338; 43550; 43563; 43622; 43743; 43771; 43826; 43833; 43878; 43884;43977; 44164; 44180; 44255; 44255; 44307; 44351; 44477; 44505; 44537;44563; 44692; 44975; 45099; 45138; 45231; 45233; 45272; 45291; 45525;45525; 45542; 45544; 45556; 45632; 45641; 45921; 45941; 46054; 46154;46154; 46171; 46182; 46284; 46356; 46518; 46786; 46897; 46916; 46916;47043; 47110; 47142; 47202; 47475; 47510; 47542; 47559; 47647; 47681;47833; 47853; 47853; 47900; 47908; 48136; 48182; 48283; 48285; 48398;48530; 48614; 48631; 48799; 48886; 48906; 49117; 49164; 49260; 49344;49352; 49404; 49464; 49509; 49518; 49591; 49630; 49665; 49710; 49860;49873; 49888; 49920; 49970; 50043; 50117; 50182; 50188; 50214; 50359;50413; 50506; 50614; 50666; 50680; 50754; 50765; 50794; 50941; 50990;51041; 51263; 51293; 51328; 51362; 51408; 51447; 51448; 51450; 51463;51532; 51543; 51640; 51666; 51666; 51702; 51737; 51740; 51746; 51814;51865; 51899; 51916; 51974; 51981; 52079; 52228; 52327; 52419; 52430;52431; 52629; 52629; 52646; 52933; 53099; 53099; 53316; 53341; 62277;66316; 66317; 66322; 66327; 66329; 66333; 66335; 66336; 66341; 66352;66354; 66369; 66377; 66390; 66391; 66400; 66406; 66407; 66420; 66423;66428; 66430; 66443; 66454; 66467; 66474; 66475; 66497; 66507; 66532;66533; 66534; 66535; 66540; 66550; 66552; 66555; 66556; 66560; 66562;66564; 66574; 66582; 66584; 66594; 66596; 66606; 66608; 66609; 66630;66663; 66685; 66686; 66686; 66694; 66697; 66711; 66714; 66718; 66721;66728; 66731; 66753; 66774; 66792; 66815; 66829; 66864; 66894; 66895;66898; 66902; 66903; 66910; 66919; 66931; 66937; 66944; 66946; 66952;66953; 66972; 66975; 66977; 66982; 67006; 67009; 67016; 67033; 67036;67037; 67055; 67069; 67070; 67074; 67075; 67654; 68103; 68225; 68977;69672; 70002; 70349; 70489; 70692; 70827; 71101; 71116; 71432; 71434;71545; 71606; 71622; 71626; 71672; 71727; 72050; 72391; 72778; 72869;73268; 73381; 73531; 73782; 74119; 74566; 74593; 75009; 75254; 75415;75923; 76362; 77133; 77391; 77533; 77577; 77577; 77636; 77728; 77805;77897; 77915; 78217; 78294; 78869; 79022; 79229; 79254; 79353; 79502;79520; 79520; 79624; 79629; 79688; 79710; 79712; 79828; 79898; 80095;80109; 80109; 80146; 80374; 80384; 80399; 80410; 80500; 80504; 80549;80708; 80772; 80910; 80915; 80924; 80946; 80948; 81129; 81289; 81315;81331; 81336; 81394; 81417; 81427; 81518; 81599; 82710; 82734; 82871;82879; 82976; 82991; 83083; 83120; 83129; 83210; 83210; 83231; 83363;83605; 84295; 84750; 84820; 84955; 85093; 85128; 85202; 85259; 85394;85394; 85497; 85509; 85561; 85624; 85634; 85678; 85682; 85690; 85805;85840; 85979; 86220; 108177; 108197; 108208; 108208; 108265; 108316;108330; 108351; 108395; 108422; 108425; 108471; 108651; 108658; 108667;108716; 108730; 108763; 108783; 108797; 108801; 108815; 108836; 109049;109089; 109108; 109123; 109153; 109179; 109200; 109221; 109265; 109269;109271; 109277; 109279; 109304; 109309; 109314; 109316; 109466; 109483;109488; 109523; 109708; 109811; 109888; 110094; 110198; 110281; 110282;110307; 110347; 110417; 110436; 110467; 110503; 110503; 110507; 110519;110578; 110582; 110585; 110653; 110703; 110741; 110746; 110772; 110788;110791; 110893; 110904; 110912; 110980; 110987; 110996; 111004; 111006;111054; 111070; 111101; 111122; 111136; 111150; 111200; 111204; 111264;111266; 111389; 111391; 111413; 111492; 111510; 111516; 111549; 111571;111634; 111693; 111705; 111714; 111721; 111722; 111750; 111755; 111765;111825; 111844; 111884; 111981; 112131; 112158; 112371; 112409; 112440;112494; 112525; 112530; 112541; 112576; 112577; 112629; 112865; 112896;112906; 113048; 113206; 113281; 113283; 113284; 113298; 113300; 113308;113394; 113399; 113431; 113488; 113538; 115111; 115143; 115155; 115223;115230; 115281; 115292; 115307; 115337; 115414; 115447; 119384; 119530;119882; 119914; 119914; 120015; 120097; 120106; 120113; 120124; 120138;120153; 120162; 120173; 120189; 120297; 120306; 120309; 120318; 120343;120362; 120375; 120383; 120413; 120516; 120533; 120544; 120551; 120561;120572; 120598; 120631; 120634; 120681; 120695; 120701; 120773; 120823;120863; 120881; 120964; 120973; 121018; 121072; 121133; 121159; 121184;121206; 121214; 121218; 121220; 121239; 121251; 121252; 121270; 121275;121316; 121341; 121355; 121365; 121412; 121415; 121420; 121458; 121459;121462; 121475; 121500; 121501; 121521; 121530; 121533; 121543; 121546;121558; 121559; 121564; 121577; 121600; 121611; 121615; 121616; 121621;121625; 121628; 121661; 121662; 121687; 121715; 121722; 121727; 121736;121756; 121770; 121776; 121792; 121798; 121803; 121808; 121828; 121877;121880; 121898; 121954; 121977; 121981; 121994; 121997; 122019; 122063;122077; 122091; 122126; 122150; 122159; 122159; 122161; 122170; 122178;122194; 122237; 122274; 122295; 122345; 122354; 122359; 122364; 122397;122428; 122443; 122636; 122684; 122702; 122762; 122787; 122796; 122822;122875; 122889; 122899; 122906; 122913; 122915; 122946; 122955; 122963;122982; 123061; 123065; 123067; 123074; 123079; 123087; 123112; 123117;123196; 123222; 123229; 123255; 123262; 123331; 123354; 123400; 123405;123408; 123425; 123433; 123436; 123439; 123441; 123448; 123459; 123474;123506; 123561; 123578; 123579; 123604; 123614; 123627; 123627; 123646;123649; 123666; 123699; 123700; 123720; 123724; 123729; 123730; 123755;123788; 123790; 123802; 123805; 123815; 123817; 123858; 123926; 123932;123952; 123980; 124009; 124014; 124020; 124042; 124043; 124052; 124059;124070; 124071; 124077; 124079; 124087; 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843133; 843134; 843139;843140; 843159; 843174; 843248; 843249; 843287; 843312; 843319; 843321;843352; 843426; 843426; 859359; 877613; 877644; 877651; 897497; 897531;897544; 897563; 897567; 897570; 897594; 897596; 897619; 897626; 897632;897636; 897642; 897646; 897652; 897655; 897667; 897669; 897670; 897673;897690; 897751; 897770; 897774; 897781; 897788; 897806; 897814; 897822;897835; 897840; 897880; 897901; 897901; 897906; 897910; 897952; 897956;897963; 897971; 897982; 897983; 897987; 897997; 898032; 898035; 898062;898073; 898092; 898095; 898096; 898108; 898109; 898122; 898123; 898138;898148; 898198; 898218; 898219; 898221; 898237; 898242; 898253; 898258;898262; 898265; 898281; 898286; 898305; 898312; 898317; 898328; 949914;949928; 949932; 949934; 949938; 949940; 950092; 950096; 950356; 950367;950369; 950430; 950445; 950445; 950482; 950489; 950574; 950578; 950607;950680; 950682; 950689; 950690; 950710; 951117; 951233; 1035182;1046522;

TABLE 6 Image ID Numbers Present in the 7K microrray. 502055; 809578;171936; 897646; 810632; 530139; 377252; 731257; 49464; 120881; 755416;950574; 712341; 783697; 434833; 258606; 743804; 39313; 144924; 209014;126401; 135450; 243638; 244310; 245489; 126219; 111634; 309515; 340974;1493527; 362624; 131839; 740554; 214816; 855521; 971372; 195330; 44351;232772; 134476; 123666; 826459; 214858; 85541; 266318; 878468; 788109;307255; 341706; 127473; 139217; 195381; 203227; 296754; 200402; 293471;243603; 809383; 322561; 292463; 725677; 839991; 428412; 796323; 399049;289978; 550355; 347373; 843067; 700792; 232826; 365973; 490778; 24918;786677; 121252; 121239; 126465; 135203; 193923; 234469; 247986; 232670;22074; 782439; 782783; 1455566; 666218; 46182; 108815; 366341; 869375;300051; 214006; 795498; 810934; 809353; 950092; 787861; 246869; 179753;430218; 433162; 812955; 36607; 115307; 127542; 137208; 195553; 194307;295723; 141171; 324861; 770887; 782503; 503841; 810617; 149737; 783696;323474; 884655; 192569; 432194; 66686; 813166; 207794; 725274; 824068;52096; 309591; 504791; 79592; 767475; 416833; 121355; 126453; 207098;548693; 244391; 244806; 281843; 141768; 427692; 21652; 1472150; 713839;727251; 79520; 877827; 549933; 285780; 240651; 769657; 46518; 525518;759873; 625923; 137535; 756931; 266106; 767471; 66467; 127185; 127943;136954; 242011; 292522; 292230; 141675; 343320; 321739; 39993; 280371;249856; 246619; 703739; 66965; 810999; 811927; 436106; 141818; 365930;360213; 841689; 897910; 824659; 45284; 345626; 415388; 813513; 293632;121365; 126508; 199571; 199641; 234419; 245217; 292559; 35236; 1031045;22260; 1492147; 40643; 208001; 826077; 491403; 310406; 1475659; 243181;68977; 345586; 243741; 79624; 773724; 1031045; 773637; 278650; 609087;813738; 246117; 120318; 127666; 136919; 295386; 207665; 295599; 211800;358531; 269815; 40304; 430614; 147414; 194949; 712683; 809507; 256907;42906; 436155; 50754; 48136; 127928; 825451; 815534; 415089; 344139;82225; 767419; 139766; 121415; 241003; 139835; 194587; 195555; 244879;132871; 149373; 323371; 22493; 1493160; 66982; 813552; 740476; 611532;757440; 1475797; 154600; 197176; 768496; 547058; 767994; 823871; 269354;611407; 854645; 345833; 665373; 207813; 126234; 130358; 136933; 195668;208383; 294167; 212180; 413633; 365841; 41199; 489506; 292475; 823562;840384; 323603; 489839; 785572; 450152; 181831; 756488; 177737; 773188;796181; 310138; 194236; 770000; 81475; 768347; 230196; 121159; 292171;142984; 200031; 246119; 244646; 247466; 42706; 501430; 23132; 1461664;813841; 148021; 753620; 855749; 153505; 1476181; 156033; 210887; 769921;84750; 80910; 711918; 109310; 430465; 120600; 625584; 66329; 244063;127173; 142326; 137663; 295600; 208524; 297437; 247635; 417226; 502506;41511; 857603; 771323; 214572; 814260; 269878; 486787; 28774; 448514;811108; 810506; 810724; 825224; 773170; 324494; 289706; 203130; 23514;138021; 108177; 121206; 151896; 138999; 292966; 213698; 296623; 248545;160664; 343073; 25584; 859478; 813630; 770858; 427812; 884641; 770880;1323539; 878564; 755054; 28098; 183337; 49630; 66718; 810059; 811024;882484; 838434; 66352; 113399; 134525; 130716; 300632; 201519; 208790;295324; 140422; 429574; 795809; 42096; 969568; 813823; 122159; 898281;810753; 882548; 42864; 815861; 377320; 810092; 194182; 814409; 83129;810264; 725335; 757489; 45645; 202901; 212236; 196214; 138374; 138837;196837; 214823; 296773; 293564; 503737; 416305; 26184; 684626; 204897;898262; 789369; 489519; 295843; 1456120; 878798; 810452; 293820; 261519;841308; 85840; 119133; 740672; 882439; 298303; 66369; 297439; 122019;130756; 140210; 200418; 229290; 239712; 140655; 504794; 487929; 42352;971199; 292306; 36387; 784224; 884842; 123264; 488964; 824552; 767638;26021; 41591; 77728; 784772; 429721; 487115; 971212; 855872; 66898;233349; 123817; 138304; 248535; 196849; 213875; 296857; 197413; 298384;502818; 815284; 742082; 271985; 843287; 756600; 415817; 1493383; 878681;41658; 209137; 123926; 68225; 951233; 782578; 429349; 432042; 897865;66390; 247710; 357970; 130791; 141106; 293128; 240050; 243460; 241113;325155; 810083; 1409509; 359747; 307231; 840889; 502690; 428103;1473304; 825418; 810600; 46897; 77897; 48631; 504774; 488645; 323500;773568; 511909; 123255; 137182; 123858; 134192; 144956; 243652; 295140;296141; 297919; 324225; 771197; 815110; 85509; 841149; 842894; 289447;344997; 461516; 1055753; 144816; 196115; 211548; 785793; 781050; 590544;377275; 773511; 66540; 111884; 122126; 130747; 141169; 201586; 292308;293356; 144878; 296095; 112565; 213607; 214068; 166199; 788647; 247103;455121; 1486082; 825606; 48614; 41356; 112629; 564803; 814526; 263836;854668; 868332; 280740; 159166; 129177; 124730; 131877; 262920; 196636;121727; 296170; 142927; 359933; 488707; 712460; 23831; 841703; 898092;452374; 757873; 487297; 45636; 23772; 247281; 201628; 839516; 758365;856354; 141852; 1031747; 270136; 66533; 121072; 123065; 134829; 140759;206781; 240318; 144885; 825842; 795735; 810923; 725746; 221092; 124127;297392; 858204; 854760; 123916; 825740; 172765; 293104; 827144; 841093;814119; 878744; 757222; 255333; 324715; 151261; 123112; 244062; 132285;244299; 201006; 300972; 296188; 130773; 809517; 299274; 815535; 809598;843159; 85497; 769890; 855624; 471725; 1358393; 70002; 308588; 86220;839094; 788421; 174627; 856174; 511459; 293191; 123788; 122237; 134368;140966; 204098; 240586; 310034; 110347; 566474; 810096; 292996; 469412;812083; 758222; 502832; 179603; 110226; 826352; 80146; 347434; 85678;144777; 137638; 755751; 897107; 866882; 593183; 66475; 120309; 124788;132358; 151418; 201030; 243808; 132464; 108330; 376767; 365177; 40704;150702; 842846; 898122; 731648; 588829; 489553; 511814; 823851; 155583;897670; 815235; 428231; 770444; 251936; 148225; 416643; 111492; 121133;126221; 134856; 141230; 203791; 240674; 292633; 111054; 324313; 810229;341328; 308437; 81417; 275180; 590727; 82131; 193087; 45556; 190468;124781; 897952; 826350; 293403; 489664; 430928; 745214; 784109; 240977;111136; 127462; 133130; 292496; 201902; 308231; 136855; 132066; 430153;795277; 66731; 812246; 430318; 85634; 878835; 241489; 1404841; 753430;229723; 43207; 897982; 795543; 448190; 205239; 471598; 586895; 784253;277274; 121462; 126371; 138550; 194704; 203805; 241350; 195911; 111264;487819; 795439; 24085; 681948; 809394; 66534; 460487; 949939; 162533;207082; 213890; 27548; 258790; 796147; 356707; 382564; 745007; 755228;788420; 142076; 110507; 127881 137653; 195387; 202066; 243385; 292812;135220; 488386; 429799; 49553; 159118; 44563; 701481; 83605; 166245;49318; 1391682; 357031; 136744; 813714; 839552; 71101; 809694; 433567;853809; 971382; 726791; 34302; 198647; 126650; 136301; 234380; 205490;245484; 308163; 194155; 269354; 366848; 1412398; 810512; 34849; 897788;360885; 145112; 287125; 183602; 813444; 823876; 137794; 51974; 897971;143306; 244951; 239446; 49560; 796475; 66902; 161195; 128193; 132748;211319; 202485; 309685; 299442; 230116; 810762; 324699; 32493; 30093;36374; 840865; 773301; 33643; 825170; 450949; 768370; 160793; 139573;824041; 786155; 488303; 431908; 383175; 271045; 233308; 121214; 126702;135853; 206949; 205085; 245556; 139558; 80384; 291974; 811028; 1412412;795178; 241474; 774502; 160664; 754406; 796287; 220851; 770452; 345858;194214; 713469; 824393; 470061; 344243; 781047; 252663; 785707; 293177;111006; 194136; 140337; 194972; 202348; 293005; 246546; 485195; 810741;268152; 246748; 127841; 28475; 628336; 307138; 855586; 266312; 451098;724588; 741067; 243580; 813266; 75254; 811168; 744917; 264646; 796759;767236; 197676; 120297; 214744; 139051; 242010; 292628; 245765; 130892;172440; 376802; 26922; 1412503; 770014; 668851; 772455; 853368; 755402;85514; 221828; 299388; 130153; 365060; 949928; 897626; 66714; 263229;198982; 869538; 767312; 232658; 234398; 128460; 193381; 195127; 210494;292690; 232628; 586698; 249949; 42993; 430297; 756452; 240518; 260200;302286; 469306; 25988; 450777; 754085; 293325; 130820; 613126; 78217;809456; 161998; 855684; 796134; 244847; 247216; 126883; 137981; 195720;232965; 245936; 135910; 180902; 66437; 27624; 1416782; 768296; 194384;609663; 345553; 68049; 1325605; 362278; 725473; 119384; 234237; 504877;897901; 810272; 415086; 51064; 869187; 768464; 248624; 233347; 128973;470232; 195117; 210501; 293500; 130777; 755093; 811097; 43129; 884743;160723; 202682; 685371; 301735; 878406; 503717; 451907; 305336; 261828;340558; 842820; 783629; 344091; 447365; 1031552; 67759; 110893; 130276;301752; 138507; 193200; 235026; 245986; 296041; 187616; 66373; 28309;1473289; 47142; 132373; 382195; 884766; 435953; 1455976; 395708; 809776;753069; 812251; 713782; 810942; 298231; 121551; 773381; 755581; 681917;127970; 203469; 130826; 154312; 212815; 210525; 293664; 132549; 782594;502496; 43338; 773367; 179500; 809639; 704290; 324437; 724932; 488956;452780; 341942; 726637; 810986; 726236; 788107; 782853; 781139; 41345;129516; 121501; 128617; 138579; 245413; 244784; 246537; 296162; 196282;323623; 28985; 1474337; 140806; 153473; 758037; 280735; 68950; 811088;110467; 204539; 781510; 784744; 417855; 739511; 1056200; 611075; 66582;144902; 206544; 130857; 138141; 206849; 210343; 293675; 274578; 136780;795442; 43550; 755474; 469345; 809901; 71672; 488276; 193736; 247483;453107; 240367; 567265; 50506; 810703; 70489; 809526; 491565; 739109;70245; 295376; 110094; 121803; 322786; 138672; 197093; 293990; 296199;796198; 219976; 504300; 30272; 592592; 128143; 682528; 487118; 80186;436062; 1472336; 757500; 771236; 549073; 125092; 897563; 842939; 795730;490763; 882488; 302632; 66596; 128204; 296883; 130972; 138189; 359411;210565; 120701; 753381; 155072; 809596; 43622; 267420; 810325; 810358;843140; 782193; 740801; 82297; 897720; 234736; 240062; 144834; 786084;826173; 320509; 262251; 67769; 280837; 110198; 123805; 128690; 138752;197776; 214848; 296334; 121420; 259587; 810992; 31143; 263716; 754600;842802; 897690; 525926; 868652; 1472698; 1472753; 146123; 754538;810444; 789204; 785744; 810502; 343871; 773137; 838149; 66608; 247660;122354; 130572; 140252; 201173; 234170; 132789; 293510; 327221; 343343;44255; 322079; 823696; 298155; 725503; 745343; 797048; 432050; 897978;725680; 131653; 323506; 52629; 760224; 770614; 510002; 306444; 842861;292232; 125828; 124132; 207649; 139189; 197736; 230180; 295501; 172721;810395; 470144; 183556; 72869; 758266; 236305; 868484; 756556; 1456937;1474684; 362853; 727210; 701112; 713886; 594743; 490556; 756596;1142132; 840766; 66630; 210923; 122359; 130582; 471372; 207016; 241475;193937; 143756; 341588; 279399; 346009; 811044; 51448; 815555; 971276;25517; 395711; 279378; 124138; 770192; 245920; 79022; 66564; 488157;969854; 505491; 842918; 211813; 128503; 124143; 294483; 288658; 292568;243405; 295206; 244055; 365665; 809488; 878652; 129506; 796000; 782811;769716; 878280; 1048985; 34184; 841357; 782800; 825433; 146577; 756709;252515; 129387; 843098; 66910; 111549; 122684; 130977; 138337; 201334;241587; 230274; 120306; 366009; 291623; 379920; 811162; 628357; 784777;461804; 756533; 769552; 756708; 471200; 123730; 134748; 739126; 40017;471859; 49963; 135085; 810801; 234036; 110578; 123932; 132848; 293755;198026; 243887; 295321; 123229; 810664; 810389; 741880; 327350; 148421;796984; 83444; 491692; 1309620; 1032405; 345348; 813675; 784124; 788745;632137; 257523; 770394; 362910; 299559; 66903; 295985; 122796; 135058;137760; 206882; 240992; 366526; 128795; 280286; 66965; 448619; 809464;127821; 244147; 431296; 869466; 263894; 814080; 26314; 299679; 80549;815285; 898258; 435551; 23012; 625683; 66977; 200863; 233688; 132524;470348; 194297; 485858; 297411; 811890; 810510; 491157; 502518; 296587;71727; 795830; 756502; 896962; 1343468; 700721; 119530; 754355; 81599;73381; 207358; 291448; 77539; 51826; 610097; 546600; 111714; 122397;135777; 137862; 204638; 241258; 345342; 130610; 795499; 810989; 366893;767851; 43826; 307532; 284001; 484874; 280252; 265102; 178463; 123980;81518; 814465; 121776; 857661; 50519; 345957; 839888; 274529; 110912;124126; 132623; 160616; 197500; 296180; 129600; 129514; 488359; 795529;810802; 124824; 504763; 774754; 462953; 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328745; 206816; 111391; 341821; 136557; 240748; 207771; 243113;469952; 232586; 129530; 795582; 359835; 66507; 824568; 341588; 361122;417920; 399604; 753457; 357220; 129644; 897544; 897774; 244955; 725308;531319; 809876; 594633; 144042; 206841; 128518; 133534; 191599; 202703;243653; 324342; 137158; 505573; 341654; 770901; 137940; 796994; 504226;810142; 236034; 868575; 382787; 248531; 138991; 47853; 788566; 268727;857264; 855385; 741885; 47481; 241988; 244154; 125589; 136706; 200015;204300; 243199; 111150; 234539; 67031; 66457; 205303; 753104; 382654;323777; 362795; 300012; 433544; 196348; 176606; 72050; 825295; 827120;66574; 41406; 770074; 845519; 813384; 47853; 341336; 128905; 133519;199327; 202931; 195487; 295889; 308415; 345523; 428773; 1410444; 753211;815501; 128302; 47359; 840753; 878112; 263097; 241160; 668442; 898328;898073; 415022; 51582; 770388; 759173; 281114; 235070; 127216; 136801;197323; 210548; 292312; 126230; 305227; 271952; 341901; 384851; 149910;276547; 897673; 357681; 725877; 75644; 148444; 810213; 184240; 70349;77391; 825478; 769846; 769537; 755952; 81203; 1046542; 243817; 196109;130053; 134495; 196125; 203400; 295044; 296562; 284592; 213660; 502762;1475633; 235934; 109708; 897781; 809828; 884546; 489677; 755663; 211216;813854; 484535; 841331; 343744; 486591; 773286; 856447; 244955; 121981;243403; 138496; 197637; 208940; 292391; 128245; 280882; 809758; 810463;461425; 666829; 208718; 950682; 429323; 970591; 73527; 345077; 40781;123802; 166236; 825585; 898312; 135247; 50930; 345935; 840466; 42627;292749; 120375; 243291; 296552; 196303; 207448; 295590; 214331; 138533;417694; 140267; 1476053; 245242; 264117; 772878; 453147; 384015; 882510;431501; 813402; 345208; 564621; 898062; 565235; 417694; 878833; 745188;859422; 204735; 121600; 210688; 139593; 194906; 208434; 293421; 129616;292726; 360075; 37196; 1358266; 770794; 240249; 788136; 343736; 264556;788205; 377468; 47900; 196501; 210317; 760298; 825296; 293715; 855438;124575; 725630; 27544; 245195; 207006; 129342; 292392; 196345; 211951;296102; 214043; 144861; 343990; 51666; 1323432; 130541; 241348; 489079;562115; 361688; 738900; 506548; 47559; 811911; 813536; 686164; 815287;782439; 884673; 725877; 594517; 108783; 121715; 127409; 144793; 194985;240138; 294311; 134312; 302369; 809824; 37451; 1404995; 296880; 44164;788518; 196543; 290724; 1468461; 378488; 668685; 183476; 549146; 782315;503617; 490232; 415870; 884993; 839374; 30850; 132630; 120383; 203514;138601; 243784; 212098; 296559; 214614; 246377; 115443; 53316; 1323203;275738; 84295; 236282; 271006; 813637; 745249; 292213; 768205; 210862;705110; 840821; 490772; 45376; 841287; 877835; 201241; 121756; 130057;139962; 195365; 137396; 293306; 136984; 309776; 771172; 38471; 1376853;809938; 813651; 587847; 236155; 361807; 1412300; 385003; 296476; 767034;810813; 824352; 488888; 148028; 502565; 813815; 109466; 120964; 131104;167076; 280122; 234080; 296602; 230613; 135713; 365536; 1456419; 39884;511521; 40017; 486221; 291985; 823665; 878130; 823590; 741815; 241412;34439; 840776; 151501; 415554; 1031185; 511091; 200934; 121792; 130107;140334; 197856; 241097; 293785; 210610; 321189; 795453; 39159; 1161797;22040; 823775; 82710; 562729; 858292; 1416502; 385017; 246786; 320606;713382; 724387; 814381; 810960; 340657; 175536; 328207; 66497; 112530;121018; 134235; 195429; 200604; 233399; 297110; 239711; 203348; 771016;32257; 141966; 810408; 739901; 855061; 856454; 1469230; 878182; 809892;31093; 184175; 785575; 840978; 454440; 435611; 460403; 741831; 66407;129331; 343352; 139837; 139462; 197907; 239835; 191572; 293457; 324815;44310; 39285; 210575; 589115; 154608; 841370; 756595; 345538; 1473131;713263; 22918; 144675; 759948; 45099; 196387; 884436; 624429; 342349;40721; 206794; 123074; 131563; 214205; 201192; 212712; 137139; 134537;233645; 782578; 1374571; 810019; 417403; 949938; 250883; 454672;1469292; 811013; 813533; 767765; 810053; 705188; 306013; 769686; 868630;60565; 77244; 193987; 241539; 124091; 130342; 140455; 198011; 230359;195132; 233299; 415178; 782537; 39884; 753418; 212496; 897906; 382693;857243; 897164; 1434905; 813983; 206217; 127519; 773319; 897751; 127925;428223; 32304; 743701; 530035; 214906; 111389; 209264; 131886; 142733;201961; 212787; 240914; 127646; 307314; 428582; 1471841; 361943; 795847;841641; 725284; 74537; 1470048; 214965; 34888; 380394; 810057; 785371;305606; 377560; 287745; 435855; 30473; 294040; 292482; 124079; 135892;191516; 232837; 244201; 246652; 281125; 205152; 144862; 782789; 667482;773618; 28218; 855547; 854138; 1388373; 814124; 173674; 768272; 898148;73531; 38763; 505005; 435076; 489823; 267865; 293901; 230247; 122899;196185; 156270; 201317; 243186; 126321; 132140; 161988; 321330; 1473300;77636; 81336; 701751; 272529; 795321; 448432; 774078; 321529; 823691;301976; 711450; 85805; 969877; 241736; 487425; 877664; 136073; 111266;128785; 130233; 204179; 199602; 245806; 201203; 161993; 214583; 771058;260303; 753775; 840364; 783849; 293925; 744417; 858469; 814287; 190887;812105; 712378; 72778; 33051; 586796; 291057; 39843; 626822; 111981;112440; 127120; 131824; 141522; 201784; 243245; 109314; 241038; 417305;782283; 1475730; 813648; 760344; 795965; 283315; 307069; 450375; 207274;28823; 823679; 44692; 767345; 156386; 46938; 624617; 796278; 773203;345090; 113284; 127415; 135303; 135789; 199709; 245899; 248478; 206867;209182; 66656; 809454; 122636; 788511; 322617; 344589; 26295; 1422723;814444; 769676; 756847; 898198; 781097; 77577; 755599; 795729; 882511;321389; 340840; 123433; 123436; 682555; 183120; 201818; 243317; 194131;234537; 269680; 810272; 151104; 810156; 826254; 491113; 489626; 856519;451706; 154651; 726779; 243343; 726147; 668182; 154093; 855487; 80649;378813; 611150; 139199; 113283; 124822; 134256; 188388; 245586; 244846;294995; 141931; 268951; 241847; 124261; 471498; 166195; 760148; 854701;277660; 1155191; 815794; 809588; 156045; 825442; 796904; 246035; 742132;866874; 858153; 1031748; 66391; 199627; 123459; 136775; 143748; 207881;242642; 124232; 208210; 195370; 810448; 486544; 45291; 950356; 269815;322160; 610362; 453689; 302190; 31842; 137836; 80948; 781704; 297061;866702; 741891; 51363; 839882; 234331; 113308; 160488; 135653; 194401;204684; 244313; 139226; 142851; 195034; 810299; 469954; 51362; 80946;191664; 362059; 611581; 187614; 182661; 341310; 810761; 814595; 109888;307933; 588915; 415529; 877832; 82556; 125741; 202209; 127099; 137020;143962; 207952; 244227; 121611; 813410; 132217; 810754; 768561; 128126;70692; 505573; 882459; 814266; 462595; 51447; 230100; 128530; 75923;824906; 139278; 285460; 416390; 781075; 839980; 133820; 110417; 130043;135710; 295741; 203287; 246478; 194399; 212542; 115408; 501674; 529861;823859; 767202; 897596; 884644; 856167; 271748; 135221; 289551; 740027;759865; 789014; 487172; 415899; 755630; 34140; 768260; 366156; 115230;127182; 144762; 202621; 208984; 245409; 296616; 142586; 415741; 346447;754998; 26811; 503097; 429555; 309893; 298965; 462645; 810504; 810010;254321; 795288; 840818; 233071; 204686; 32299; 841396; 754378; 144747;128054; 130005; 135010; 195853; 203388; 245273; 206786; 240430; 272183;503033

The disclosed microarrays can be constructed using the Image ID numberprovided in these tables, and other available information. Inparticular, the Image ID number can be searched on the ATCC website,with hypertext links available to ATCC deposit numbers for depositedclones corresponding to the Image ID numbers. Hypertext links are alsoavailable to Genbank entries, which disclose sequence information aboutthe nucleic acid at each array site corresponding to Image ID numbers.

Tables 7 and 8 summarize the results for exposure of cells to ionizingradiation using the 1.2K, 5K and 7K microarrays. Table 7 shows theresults of different radiation doses (2 and 20 Gy), and hypoxia (whichtriggers p53 induction) using the 1.2K and 5K microarrays. As shown inTable 7, a very large fraction of the genes tested showed alteredexpression. As shown in the last two rows of Table 7, a substantialnumber of genes responded both acutely (3 hours) and at a later time (24hours) after irradiation. The genes which showed expression at 24 hourswere considered particularly suitable for a clinical biodetector thatwould detect the effects of radiation exposure during a time period whenlaboratory investigations of potential radiation exposure are likely tooccur. However the genes which respond acutely (at three hours) couldalso be placed in an array to measure even more immediate exposures.Other arrays that include both acute (e.g. three hours) and later (e.g.24 hours) response can be included in a single array. Differencesbetween response of these two subsets can be used to help determine aprobable time of radiation exposure. For example, if the three hourresponders are positive but the 24 hours responders are not, then thetime of exposure would have been at least three but less than 24 hoursbefore the test. The results shown in Table 8 focus on lower doses andlonger timepoints than the studies shown in Table 7. TABLE 7 Summary ofresults for microarray analysis of stress responses¹ Array² TargetsTreatment Induced³ Reduced⁴ 99% Conf.⁵ 1.2K 1238 20 Gy + 4 hr 38 110.63-2.37 Chip 1238 6 hr hypoxia 135 19 0.51-2.47 1238 24 hr hypoxia 2028 0.55-2.38   5K 5408 2 Gy + 3 hr 67 21 0.68-2.31 Chip 5408 2 Gy + 24 hr161 26 0.69-1.89¹Results are summarized for microarray hybridizations conducted in ML-1cells. Many of the results for row 1 and 3 have been verified byquantitative single-probe hybridization.²Two different microarrays (chips) used contained only limited overlapin the genes represented.³The number induced refers to cDNA clones showing significant induction(>99% confidence).⁴For cDNA clones showing a significant reduction, values are shown forthose having at least a 2-fold reduction in expression compared tountreated cells; all these values exceeded 99% confidence.⁵Values represent range of relative expression of irradiated samplecompared to untreated control; e.g. only targets showing an increase inthe relative mRNA level of 2.37-fold or more were scored as induced inthe first row.

TABLE 8 Summary of γ-ray Experiments with 7K Microarrays Cell Line DoseTime # Up^(a) # Down^(b) 99% Conf. C.V. ML-1  10 Gy  4 hr 86 23 .38-3.00.16 ML-1   2 Gy  4 hr 32 3 .51-4.38 .27 ML-1 2.5 Gy 24 hr 62 8 .93-7.08.23 ML-1 2.5 Gy 48 hr 60 17 .60-4.85 .24 ML-1 0.2 Gy  3 hr 86 29.74-2.43 .15 ML-1 0.2 Gy 24 hr 114 11 .83-3.42 .18 ML-1 0.02 Gy   3 hr68 40 1.16-9.18  .24 ML-1 0.02 Gy  24 hr 55 6 .78-5.92 .23 HL60 0.25 Gy  3 hr 77 2 .63-4.95 .24^(a)Number of Image ID clones that were upregulated in response theirradition.^(b)Number of Image ID clones that were downregulated in response theirradition.

Tables 9-12 lists the clones (by Image ID number) for which there was99.9% confidence that the genes were differentially expressed in ML-1cells with the 7K array at: 24 hours after 200 cGy of irradiation (Table9); 24 hours after 20 cGy of irradiation (Table 10); 24 hours after 2cGy of irradiation (Table 11) and; 48 hours after 200 cGy of irradiation(Table 12). Late responding genes were found by using RNA harvested atthe timepoint of interest, for example 24 or 48 hours after exposure toionizing radiation. A more detailed timecourse of expression (such asthat disclosed in EXAMPLE 3) can be used to determine if expressionpeaks at the time of identification (24 or 48 hours), or if there is asustained elevation of expression. To identify genes for othertimepoints post-exposure, or for different exposures, the cells would betreated with the agent/dose of interest, and the RNA would be harvestedat the appropriate time. TABLE 9 Image ID numbers of genesdifferentially expressed after 200 cGy and 24 hours. 280386; 753418;308588; 241412; 109123; 47475; 549146; 841278; 221846; 159455; 251516;345839; 240249; 136114; 703846; 66972; 153025; 713145; 366558; 209340;242780; 293325; 843312; 240843; 35828; 810391; 264117; 293940; 244227;262231; 180520; 294487; 562927; 160793; 72391; 120362; 346995; 232837;297442; 295939; 810372; 120572; 666658; 550355; 48398; 469954; 110653;211548; 260619; 128290; 210522; 241788; 122091; 140921; 120881; 755416;504226; 738899; 346552; 248256; 66728; 122159; 232612; 753447; 813591;245585; 325375; 289551; 366341; 36950; 236355; 125092; 204814; 196387;843287; 34773; 120695; 287300; 234376; 813179; 293557; 712840; 244703;24415; 138579; 229723; 206816; 108658; 157856; 308041; 727026; 292217;231675; 135240; 194965; 246661; 812196; 135538; 41452; 123474; 49710;823928; 141818; 823940; 327094; 141854; 282051; 195720; 813675; 795543;128530; 124071; 810156; 293727; 768638; 773332; 121159; 724892; 295710;809588; 121251; 139835; 781341; 815534; 770355; 841703; 293306; 202549;39993; 195365

TABLE 10 Image ID numbers of genes differentially expressed after 20 cGyand 24 hours. 854401; 854701; 131887; 112896; 296149; 725501; 41648;66400; 132690; 415022; 770866; 292222; 323917; 83210; 772220; 288896;110436; 248306; 743278; 826984; 760148; 322160; 234527; 969748; 291985;795877; 430968; 487172; 198815; 343646; 204814; 562115; 491692; 274638;244227; 111571; 242780; 279195; 509887; 343744; 769603; 264117; 771295;322961; 825295; 141768; 753418; 686164; 383016; 109123; 80649; 841282;450375; 66898; 111693; 210317; 627251; 24415; 740027; 685801; 448619;773567; 768272; 854576; 510381; 66457; 298963; 611443; 243100; 771023;884500; 221846; 200031; 843312; 839516; 120695; 195129; 266094; 234562;127709; 704690; 768443; 796278; 242087; 47475; 195346; 123441; 140921;214165; 34255; 549146; 626555; 713193; 142087; 811015; 815816; 126453;123408; 46786; 359395; 269381; 122443; 1408710; 825442; 111765; 241412;490763; 739183; 30272; 152453; 108801; 82556; 204148

TABLE 11 Image ID numbers of genes differentially expressed after 2 cGyand 24 hours. 703846; 84820; 745347; 274638; 24415; 841278; 66975;221846; 712840; 308041; 562927; 359184; 135538; 308588; 282051; 738899;109123; Bcl-x; 359793; 246661; 204814; 755416; 897781; 773332; 813591;52681; 810142;

TABLE 12 Image ID numbers of genes differentially expressed after 200cGy and 48 hours. 264117; 240430; 811015; 262231; 308588; 159455;549933; 323474; 840944; 432194; 109123; 321739; 366945; 843287; 240249;111765; 48182; 130541; 771295; 624627; 294487; 812126; 345559; 382654;755037; 203130; 193087; 843312; 214133; 811000; 3084437; 431655; 448619;823940; 41356; 612616; 725746; 47475; 549146; 162775; 35828; 46284;120881; 770935; 208161; 745188; 843139; 377671; 838373; 809639; 686164;206816; 770394; 179617; 809394; 591281; 294496; 124795; 782760; 767828;308041; 731308;

Genes with sustained elevated expression (for example 24-48 hoursfollowing the exposure) may be suitable for inclusion in probe sets forclinical tests which would not be performed for several days afterpotential exposure to ionizing radiation. Using dose responseinformation, different probe sets can be designed for different clinicalsituations, tailored to detection of exposure a certain number of hoursfollowing potential exposure.

Probe sets can also be designed that detect certain subsets of genesthat are differentially expressed at a particular post-exposure time andlevel of expression. The levels of differential expression can becorrelated to the dose of ionizing radiation to which the subject wasexposed, such that the probe set can also be informative about the doseof exposure. This information can provide helpful prognositcinformation, such as the likelihood of carcinogenesis brought about bythe exposure. Alternatively, levels of differential expression can beused to determine a subjects' response to radiation therapy for a tumor,as described in EXAMPLE 10.

EXAMPLE 7

Nucleic Acid Probe Sets

Nucleic acid hybridization technologies may be used to survey geneexpression patterns in organisms or cells that have been exposed toionizing radiation. Such technologies are not necessarily limited tonucleic acid arrays. By way of example, northern blot and/or dot blottechniques (see EXAMPLE 3) may also be used to determine thequantitative and qualitative expression patterns of some or all of thedisclosed radiation responsive sequences.

While more conventional nucleic acid hybridization techniques (such asnorthern and dot blots) have been used for many years, nucleic acidarray technology is now widely used for monitoring and analyzing geneexpression patterns. This array technology may be used in a number offorms, including microarrays. Microarrays typically comprise a largenumber of nucleic acid probes spotted at high density onto a surface.Descriptions of nucleic acid array and microarray technology may befound in the scientific literature, including, for example, in Chee etal., Science 274:610-4 (1996); Lockhart et al., Nature Biotechnol.14:1675-80 (1996); Lipshutz et al. Biotechniques 19:4427 (1995);Southern et al., Trends Genetics 12:110-5 (1996); Soares et al., Curr.Op. in Biotech. 8:542-6 (1997); Ramsay et al., Nature Biotech. 16:40-4(1997); Schena et al., Science 270:467-70 (1995); Schena et al.,BioEssays 18:427-31 (1996); DeRisi et al., Science 278:680 (1998), andIyer et al., Science 283:83-7 (1999). Detailed technical descriptions ofvarious forms of this technology can also be found in the patentliterature, including in the following patent documents:

U.S. Pat. No. 5,744,305 (“Arrays of materials attached to a substrate”);

U.S. Pat. No. 5,807,522 (“Methods for fabricating microarrays ofbiological samples”);

U.S. Pat. No. 5,545,531 (“Methods for making a device for concurrentlyprocessing multiple biological chip assays”);

U.S. Pat. No. 5,593,839 (“Computer-aided engineering system for designof sequence arrays and lithographic masks”);

U.S. Pat. No. 5,837,832 (“Arrays of nucleic acid probes on biologicalchips”);

WIPO publication number WO 9710365 (“Expression monitoring byhybridization to high density oligonucleotide arrays”).

cDNA arrays can be formed on non-porous surfaces (such as glass) by insitu synthesis of oligodeoxynucleotides on a chemically sensitized glasssurface (as in WO 92/10588 and WO 95/11995), or by roboticmicropipetting of nanoliter quantities of DNA to predetermined positionson a non-porous glass surface (as in Schema et al., Science 270:467-470,1995, and WO 95/35505). DNA may be coupled to the solid support byelectrostatic interactions with a coating film of a polycationic polymersuch as poly-L-lysine (WO 95/35505), or covalently bound to the solidsupport.

Nucleic acid arrays employ conventional nucleic acid hybridizationmethods that have been used for decades to identify and quantify nucleicacids in biological samples (such conventional methods include southernand northern blots, colony hybridizations and dot blots). However,whereas such conventional techniques typically employ one or twohybridization probes to obtain information on the expression patterns ofone or a few genes, array techniques typically employ very large probesets (for example at least 100, 1000 or even 5000 or 10,000 probes) toobtain data on the expression of a vast number of genes simultaneously.

The basic principle underlying the array technology is the hybridizationof a sample nucleic acid composition with a defined set of nucleic acidprobes, followed by detection of specific hybridization of the sample toone of more of these probes. The hybridization pattern obtained is thenanalyzed and compared to hybridization patterns obtained with controlnucleic acid samples.

Most arrays comprise a defined set of nucleic acid probes immobilized ona fixed surface in an ordered and known sequence, forming an array ofdiscrete spots of nucleic acid material. A number of substrates may beused to form the fixed surface, including silica-based chips, nylonmembranes, microtiter plates and glass slides. Each probe within the setis typically produced by polymerase chain reaction amplification(alternative methods include purification from cloning vectors and, foroligonucleotide probes, chemical synthesis). Each amplification product(probe) is then typically spotted onto the fixed surface using amechanized means (such as a robotic arm) to form the array. The samplenucleic acid composition (for example, labeled cDNA produced by RT-PCRfrom mRNA extracted from a tissue sample) is labeled with a detectablemarker (e.g., a fluorescent label) prior to hybridization to the arrayto permit detection of specific hybridization of the sample to aparticular probe. Following hybridization, the hybridization pattern isdetected and an image of the array is produced for analysis of geneexpression. The use of multiple different fluorescent labels to labelthe samples permits multiple different samples to be hybridized to asingle array. Typically however, no more than two samples are hybridizedto a single array.

Alternatively, the arrangement may be reversed. The fixed array mayinclude a number of nucleic acid test samples and the probes may behybridized to a number of duplicates of the array. In this situation, alarge number of test sample nucleic acid compositions are attached tothe surface to form the array. Subsets of the nucleic acid probe set aresubsequently hybridized to duplicates of the array, and hybridization ofthe probes to individual spots of the array is detected. The use ofmultiple different fluorophores to label the probes permits multipledifferent probes from the probe set to be hybridized to a single array.

By way of illustration, a microarray may be produced and employed usingthe following techniques:

Each probe in the microarray probe set is produced by PCR amplification.This may be achieved by using primers that are specific for the sequenceof the differentially expressed gene (as disclosed herein) inconjunction with genomic DNA or cDNA as a template. The amplified PCRproducts are purified to remove excess primer and template, for exampleby column chromatography, or by simple sodium acetate or ammoniumacetate precipitation, followed by either isopropanol or ethanolwashing, and drying. In certain instances, the probe may be used withoutpurification. Following purification, the PCR-amplified probes areresuspended in 10 μl of a salt solution (3×SSC) and transferred into96-well or 384-well microtiter plates (if they were not already in amicrotiter plate from previous steps). The samples are mechanicallyspotted onto coated glass slides, using a robotic arm containing pinsthat transfer an amount of each probe from the microtiter plate onto thecoated glass slide. Several hundred or several thousand probe spots arespotted onto the array such that each probe is represented by a discretespot with a center to center distance between spots of about 250 μm toabout 500 μm. The glass slides are then processed to cross-link theprobe set onto the glass surface.

The nucleic acid sample to be hybridized to the array is typically cDNAobtained by RT-PCR of mRNA extracted from a biological sample (e.g.,plant or animal cells) (see EXAMPLE 2). The detectable label (e.g., thefluorophore) may be incorporated during the RT-PCR amplification step.Typical experiments involve either single-color fluorescencehybridization to measure the absolute levels of gene expression in asingle sample, or two-color fluorescence hybridization to examine therelative expression of genes in two different samples.

For single-color fluorescence hybridization experiments, mRNA isisolated from a sample of interest and used as a template to producecDNA. The cDNA is labeled, for example using a fluorescent dye such asCy-3 or Cy-5 (Amersham Pharmacia Biotech, Piscataway, N.J.), or anyother fluorophore or label. The label can be incorporated directlyduring the reverse transcription step. The sample is then hybridized tothe array. Following washing to remove non-specifically bound sample,the array is scanned for fluorescent emission following laserexcitation, and the intensity of each fluorescing spot is measured. Theintensity of each spot is approximately proportional to the expressionof the gene (corresponding to the probe) in the sample examined. Thisdata provides an indication of the expression of a particular gene inthe tissues from which the sample nucleic acid was prepared.

For two-color fluorescence hybridization experiments, RNA is isolatedfrom two samples of interest and labeled as described above, except thateach sample is labeled with a different fluorescent label, each of whichfluoresces at a different wavelength (for example, one sample may belabeled with Cy-3 and the other with Cy-5). After the two samplepreparations are labeled, they are mixed together and hybridized to asingle microarray. After washing, the microarray slide is scanned in twofluorescence channels. Because the two fluorescent labels are selectedsuch that their emission spectra do not overlap, the signal of each ofthe two fluors can measured for each of the probes of the array. Theabsolute levels of intensity for each probe in an array is approximatelyproportional to the expression of the gene in the sample examined, andthe ratio of the two fluor intensities indicates the relative expressionof a gene in the two different samples.

Each probe includes at least one copy (and more typically many copies)of an isolated nucleic acid molecule. Typically, the nucleic acidmolecule is in substantially pure form, i.e., while there may be smallamounts of other nucleic acid molecules in the probe preparation (suchas degradation products), the selected nucleic acid molecule will be thepredominant nucleic acid molecule present. In addition, a probe isgenerally of known sequence. A “probe set” comprises a collection of twoor more such probes (the individual probes within the probe set notbeing co-mingled). A probe set will include probes selected for aparticular purpose (such as detection of acute or semi-acute exposure toionizing radiation), but may also include controls or other probes fordifferent purposes. In some examples, at least 10% of the probes in theprobe set are selected for the particular purpose of determining anactual or potential biological response to an actual or potentialradiation exposure. In other examples, at least 5, 10 25, or 50 of theprobes of an array are selected for this purpose.

A hybridization probe for use in an array produced according to thepresent invention may be referred to as a sequence “representing” aparticular gene product. A sequence “representing” a particular geneproduct is one that will specifically hybridize to a nucleic acidmolecule encoding that gene product, thereby permitting identificationof that gene product. A sequence representing a particular gene productmay comprise an entire cDNA sequence (or the corresponding genomic genesequence) or less than an entire cDNA sequence. For example, the probemay comprise an oligonucleotide comprising a minimum specified number ofconsecutive bases of a selected gene that is differentially expressedfollowing irradiation. Oligonucleotides as short as 8-10 consecutivebases of a cDNA will be effective to produce meaningful gene expressiondata using microarray technology. For example, a nine baseoligonucleotide can distinguish 262, 144 transcripts (4⁹). However, forenhanced specificity of hybridization, longer oligonucleotides may beemployed, such as at least 10, 15, 20, 25, 30, 50, 50 or moreconsecutive bases of a cDNA. Furthermore, a probe “representing” aparticular gene product need not be a complete match. While probes thatshare 100% sequence identity over their entire length to thecorresponding cDNA sequence will typically provide enhanced specificityof hybridization, probes that share less than 100% sequence identity mayalso be useful in such microarray applications. Typically, such probeswill share at least 70% sequence identity with the corresponding cDNA,but probes sharing at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, and 99%sequence identity may be utilized to achieve enhanced specificity.

EXAMPLE 8 Examples of Arrays for Detecting Genotoxic Stress

An examples of a probe sets that has been used to make a cDNA array thatdetects differential expression of genes following irradiation is shownin Tables 13. The clones on this chips include all the positive hitsfrom studies with larger arrays (5K and 7K), in addition to probes forother genes that have been reported in the literature as beingstress-induced. TABLE 13 Image ID Numbers of Genes DifferentiallyExpressed in Response to Irradiation. 21652; 23132; 753430; 78294;127193; 155287; 825080; 703846; 121877; 128785; 549933; 781139; 811927;884500; 381287; 700792; 196185; 133225; 739183; 502832; 40692; 626531;840511; 39993; 41511; 812196; 346995; 123926; 240249; 504226; 510760;160573; 324861; 81203; 85195; Bcl-x; 784179; 66428; 125741; 134537;795877; 46284; 134476; 377587; 141763; 22260; 25584; 753418; 141818;129506; 547058; 814701; 686172; 121159; 297442; 289606; 78353; 34184;785847; 760299; 788185; 134235; 143208; 323474; 433253; 950507; 754378;126858; 40304; 42096; 727026; 229723; 51448; 191664; 203132; 769846;240961; 49410; 80162; GADD153; 322160; 77636; 241539; 136984; 856961;41356; 511066; 323577; 25588; 22493; 26184; 345839; 130788; 120572;549146; 772220; 77728; 247216; 143209; 756405; 624617; 878676; 12637;41074; 33051; 131886; 138496; 811166; 757873; 509887; 108801; 810372;41199; 42352; 140716; 550335; 125092; 788141; 297392; 294487; 160664;795893; 147050; 22428; GADD34; 293925; 80946; 201241; 77087; 22293;740027; 840691; 770935; 26922; 28985; 36393; 214537; 50506; 562927;72391; 843312; 135240; 138579; 45578; 243882; 504745; CR6; 297061;257445; 137020; 234527; 431655; 366945; 120383; 138477; 772878; 42993;43550; 160793; 221846; 714106; 841703; 859339; 841278; 272183; 504372;28985; 291985; 279172; 206651; 111004; 31169; 50794; 773319; 686164;245774; 27624; 30272; 813614; 359184; 308041; 66728; 774036; 826166136114; 140921; 266094; 882484; BAX; GADD45; 300137; 151501; 139462;198011; 856519; 247483; 131010; 204179; 824352; 43129; 43622; 289551;135538; 95036; 842802; 214162; 122822; 487819; 502977; 36950; 53164;384015; 454672; 230247; 113284; 489506; 550353; 843249; 815245; 120306;28309; 31143; 204299; 813841; 823928; 306901; 293940; 781019; 154214;157856; 235938; 745116; c-Myc; MDM2; 490232; 769686; 197720; 230191;756549; 487348; 133331; 296189; 66975; 43338; 44255; 666658; 261828;24415; 841334; 292217; 245444; 119133; 41452; 110589; 223350; 1388373;112530; 111693; 489489; 727551; 231355; 796904; 590544; 31210; 34357;128143; 589115; 156473; 753104; 739625; 587847; 194965; 240843; 884719;781017; RelB; 417694; 204686; 744047; 204122; 229617; 179617; 814123;244386; 132304; 855521; 44537; 47833; 159455; 145503; 785574; 192694;205049; 282051; 323185; 491403; 35516; 193901; 878182; HsKg21F11;296393; 123436; 767828; 49509; 74566; 856489; 85195; 32898; 34396;34773; 153025; 809939; 770957; 781341; 23073; 232837; 242780; 810999;128493; V(D)J (RAG2); 270626; 340657; 1031185; 208940; 295497; 432194;214133; 193182; 135713; 253009; 45941; 50117; 308588; 175123; 789182;768638; 295939; 50765; 282978; 884425; 234357; 280507; 1049030; 754601;207006; 124168; 45632; 810408; 235155; 21899; 85906; 33632; 35483;738899; 327676; 232612; 753447; 48398; 231675; 204814; 260619; 251591;436062; Hbc1W8; 44495; 148028; 435855; 233939; 230359; 713236; 240634;132140; 321271; 949971; 47110; 50666; 813675; 202535; 70692; 786067;245585; 66972; 280893; 590544; 773367; 774078; 234559; 121715; 124091;308437; 49352; 429574; 50888; 44495; 36950; 39093; 813179; 725680;741769; 42373; 760224; 841641; 280386; 139226; 724888; 360778; 510130;H36; 840466; 784065; 241097; 299815; 810331; 592359; 377587; CIP1/WAF1;51293; 26578; 810391; 725266; 789369; 34689; 66430; 487777; 586839;626358; 31093; 759948; 130053; 139837; 712641; 51041; 280356; 852829;37366; 39173; 810485; 729942; 195712; 264117; 630013; 204301; 293727;296030; 193736; 740907; 433551; 52753; 839374; 82556; 295303; 112371;190887; 361097; 323577; GADD45; 51740; 244703; 128530; 592540; 287300;246722; 546600; 780947; 145383; 269606; 35318; 812105; 825295; 128695;137456; 166195; 796994; 866874; 85313; 37904; 39796; 810600; 42739;249688; 122091; 842849; 841470; 345751; 172721; 755923; 378813; 52681;51699; 252382; 27544; 246652; 121798; 28823; 51737; 770935; 145503;129865; 207794; 772878; 712840; 295710; 108351; 462953; 855521; 73527;489485; 40056; 484535; 130057; 130617; 809394; 842860; 857002; 854899;22012; 24145; 755145; 124261; 293916; 823901; 898062; 84820; 120695;125589; 310406; 377692; 220851; 951142; 795771; 305606; 131824; 134643;307532; 845415; 743230; 785778; 36393; 40026; 42059; 823940; 244767;211548; 122159; 713145; 358531; 121251; 417226; 125187; 85195;CIP1/WAF1; 236155; 233721; 66574; 295514; 428582; 35077; 241880; 161988;325062; 22293; 25725; 812965; 666218; 124824; 752732; 843321; 815539;123439; 130043; 491763; 345525; 825740; 124143; 814117; 196387; 131563;134256; 810711; 271006; 416316; 242706; 214537; 40360; 42214; 141763;120881; 274638; 701112; 172440; 199180; 123229; 268727; 309288; 26997;GADD45; 755750; 141966; 292482; 262053; 121611; 129146; 770212; 310894;159455; 23019; 26366; 180520; 205185; 669443; 177737; 824352; 613126;124071; 325375; 487820; 845363; 488596; 549139; 194600; 132708; 137016;361323; 858292; 811000; 125767; 223483; 41452; 42576; 110653; 325062;811942; 840511; 85840; 745347; 232714; 269815; 83999; 50615; 429555;854701; 28218; 200934; 427692; 30664; 234562; 898198; 810448; 70692;27549; 29054; 126858; 245296; 323506; 197525; 71545; 416833; 234376;139957; 133637; 745433; 814476; GADD153; 233071; 377560; 195429; 197907;841221; 197657; 123067; 262920; 712840; 43021; 43563; 809588; 210522;898138; 825470; 48285; 133114; 782712; 34005; 47359; 714213; 109271;128602; 40360; 340630; 773254; 843139; 172721; 27848; 30664; 26616;469954; 136235; 236355; 724892; 897690; 144762; 137853; 770424; 384081;Bcl-x; Killer/DR5; 487172; 454440; 195853; 200780; 322914; 1472735;137918; 212712; 613126; 43231; 44180; 769921; 293325; 208161; 612274;196387; 141627; 810059; 782545; 39993; 85906; 361688; 1473274; 109466;113283; 450745; 810017; 757961; 73531; 769846; 28410; 31169; 203275;810372; 137940; 241788; 795543; 815534; 139835; 195720; 266106; 253009;CIP1/WAF1; MyD118; 215000; 755599; 198593; 234484; 856167; 1320746;139490; 300866; 345751; 43241; 44307; 292806; 25588; 841149; 726086;366341; 108658; 809585; 489805; 26474; 26295; 1416502; 110582; 296472;814780; 178779; 684661; 787861; 845477; 32875; 34355; 770670; 223483;137017; 35828; 85259; 376875; 195365; 240648; 796278; 26568; V(D)J(RAG-1); 347586; 46938; 755630; 245586; 212098; 385003; 163174; 359247;141931; 491763; 45233; 49888; 823614; 212198; 74119; 82991; 120362;293557; 324494; 289428; 49950; 288650; 687054; 173674; 196109; 123459;812126; 665774; 591281; 447568; 80162; 3352; 34945; 162775; 770355;241412; 810156; 135083; 840944; 109123; 244227; 263014; 415817; Bak;43021; 415870; 460403; 208984; 214826; 1472753; 753457; 194131; 809995;251591; 46171; 50359; 727210; 248454; 361974; 796646; 141854; 248256;342008; 364510; 486074; 768064; 824081; 230100; 121600; 124822; 35185;51865; 122150; 435858; 878676; 34255; 36775; 22040; 122428; 22411;26811; 39274; 950680; 209340; 293858; 39722; 491692; GADD34; 45794;32304; 192271; 230013; 244201; 825726; 380797; 109483; 810154; 85195;47202; 50941; 135692; 135527; 77915; 49710; 246661; 129585; 204569;624627; 359793; 1467195; 1493160; 341310 244686; 126568; 49509; 43826;429494; 773637; Bcl-x; 37196; 39159; 240151; 346552; 292463; 771196;666425; 839736; 293306; 202549; 949971; 66599; 503724; 133637; 297061;773579; 239835; 194399; 511832;767765; 161988; GADD153; 51666; 123474;823691; 897781; 843287; 205993; 111981; 845477; 810142; 120468; 882510;213136; 485989; 129332; 130342; 950430; 503097; 770394; 268946; 37451;39285; 126243; 210887; 85561; 245388; 51666; 898286; 128290; 245774;788764; 245990; 592485; 264937; 594633; 626822; 123729; 50188; 204214;310894; GADD34; 53316; 131653; 755416; 810131; 897570; 327094; 206816;756502; 1035889; 454339; 82297; 236333; 80948; 135892; 130876; 841384;705274; 415529; 505491; 38471; 39884; 127677; 232772; 28475; 179403;838373; 725454; 251516; 120306; 342181; 757350; B1; 280386; 771295;136188; 297405; 296616; 31842; 51737; 810448; 47475; 813591; 773332;366728; 66975; 110746; 359661; 586796; 75644; 858293; 810813; 759865;130107; 131668; 612616; 724615; 376785; 856535

A microarray was assembled which contained each clone in Table 13printed only once on the array. However the arrays can routinely includeduplicates of each position as the stress-array, to provide anadditional degree of certainty for positive “hits.” In addition,internal controls of the type known in the art can be used on eacharray.

The probe set for a cDNA array can be made using a probe set comprisingnucleic acid molecules representing a portion of the cDNAs disclosed inTables 9-13. Alternatively, such probe sets may be used to analyze geneexpression patterns using other hybridization techniques, such asnorthern and dot blots (see EXAMPLE 3). However, useful information mayalso be obtained using arrays or other techniques that employ probe setsrepresenting less than all of the disclosed cDNAs in the array of Tables9-13. For example, arrays that employ probe sets representing between10% and 99% of the disclosed cDNA sequences may be employed. Thus,arrays employing probe sets representing at least 25%, 50%, 80% or 90%of the disclosed cDNAs depicted in any of Tables 9-13 may be employed.Alternatively the probe set may represent at least 5, 10, 25, or 50 ofthe disclosed cDNAs depicted in any of Tables 9-13. Each probe can“represent” the cDNA by having sufficient contiguous nucleotide (orvariant thereof) that hybridizes to a target nucleic acid of interest.Alternatively, an array can be made using probes that detectdifferential expression of genes within a certain time period. Anexample would be an array consisting of the probes of any of Tables 9-12or combinations thereof.

The sets of disclosed probes may also be used in nucleic acidhybridization techniques. Such probe sets are particularly useful forassessing differential gene expression in cells that have been exposedto radiation. The probe sets comprise nucleic acid probes representingspecified percentages of the differentially expressed cDNAs disclosedherein. These probe sets may be used with any nucleic acid hybridizationtechnique that can be used to analyze patterns of gene expression,including but not limited to: northern blotting, dot blotting, arraysand microarrays.

In addition, the invention also provides methods for analyzingexpression of multiple genes in a biological sample. A biological sampleis provided which includes nucleic acids, and the biological sample ishybridized to a nucleic acid probe set arrayed on a surface. Typically,the nucleic acids of the biological sample are labeled with a detectablelabel such as a fluorescent label, to permit easy detection ofhybridization between the sample and a probe. The nucleic acid probe setarrayed on the surface includes nucleic acid probes representing, forexample at least 2%, 5%, 10%, 25%, 50% or more of the radiation inducedcDNA sequences disclosed in any of Tables 9-13, such as subsets ofprobes for genes that have sustained expression at 24 or 48 hours, orexpression at a level that corresponds to clinical exposure to a certaindose of ionizing radiation.

The present invention also includes providing a plurality of duplicatearrays of test sample nucleic acid compositions (such as samples takenfrom one or more subjects) immobilized on a fixed surface. These arraysare then hybridized with a nucleic acid probe set (typically labeledwith a detectable label). Hybridization using the complete set of probesis typically achieved by hybridizing a small subset of the probes(typically 2-5 probes) to one of the array duplicates, and thenrepeating the procedure by hybridizing additional subsets of the probeset to additional duplicates of the array. To facilitate detection ofhybridization, each of the probes within a particular subset istypically conjugated to a different detectable label. The probe set usedin this method comprises nucleic acid probes representing at least 2%,5%, 10%, 25%, 50% or more of the cDNA sequences disclosed in any ofTables 9-13, or the complete probe sets disclosed in those Tables, othersubsets thereof (such as at least 5, 10, 25, or 50 of the disclosedsequences), or probe sets that are larger than any of these sets andinclude additional informative markers that have been or are discovered.

Arrays of polynucleotide DNA probes immobilized on solid supports havebeen used to study the composition of complex mixtures of DNA byhybridization techniques. For example, a complex mixture of labeled cDNAis hybridized to the DNA array under conditions of appropriatestringency, and unbound material is washed away. The array is thenscanned using a detector, such as a scanning fluorescent microscope,capable of sensing the remaining bound labeled cDNA. The intensity ofthe detected signal at any given element is a measure of theconcentration of the corresponding cDNA in the original complex mixture.See Schema et al., Science 270:467-470, 1995 and WO 96/17958.

EXAMPLE 9 Analysis of Biological Specimens

This example demonstrates that cDNA microarrays can be used to assessthe effects on gene expression of irradiating isolated human peripheralblood lymphocytes or alternatively can detect altered gene expression toassess potential radiation exposure. Peripheral blood lymphocytesprovide an easily accessible source of radiation sensitive tissue from asubject who has potentially been exposed to ionizing radiation. Todetermine whether the subject has experienced a biologically significantradiation exposure, approximately 30 ml may be obtained by conventionalphlebotomy. This amount of blood provides sufficient mRNA forhybridization to a large array (such as a 5K array or 7K array), orseveral smaller arrays (such as 1.2K arrays), during efforts todetermine which genes are differentially expressed. However, muchsmaller probe sets can be used diagnostically, with correspondingsmaller amounts of blood required.

Although this example describes methods which were used to analyzeperipheral blood lymphocytes, one skilled in the art will recognize thatsimilar methods can be used to analyze any biological specimen.Biological specimens can be obtained from subjects who have potentiallybeen, or are known to have been, exposed to ionizing radiation. Avariety of biological specimens can be used in the present invention.Examples include, but are not limited to: peripheral blood, urine,saliva, tissue biopsy, surgical specimen, amniocentesis samples andautopsy material.

Blood may be drawn from potentially exposed individuals, RNA extractedimmediately, and tested against an established baseline to determinerelative levels of a number of genes, then compared to profiles forvarious qualities or quantities of radiation, or other environmentalagents, to determine likelihood of exposure. Approximate dose and timesince exposure can also be determined.

Human blood from normal healthy donors were obtained from the NIH bloodbank (Department of Transfusion Medicine) and within 30-60 minutes ofdrawing, the components were separated by centrifugation on a Lymphoprep(Nycomed, Oslo, Norway) density gradient according to the manufacturer'sinstructions. The buffy coat layers were recovered, washed in phosphatebuffered saline and resuspended at a density of 0.5-1×10⁶ cells per mlin RPMI 1640 medium supplemented with 10% heat-inactivated (56° C. for45 minutes) fetal calf serum and 100 U/ml penicillin and 100 μg/mlstreptomycin at 37° C. in a humidified, 5% CO₂ atmosphere. Peripheralblood lymphocytes (PBLs) were allowed to equilibrate to cultureconditions for 45-60 minutes, then irradiated at approximately 60cGy/min. to total doses of 20-200 cGy using a Mark 1-68 ¹³⁷CS source(J.L. Shepherd and Associates, Inc., San Fernando, Calif.) with leadattenuators in place.

RNA was harvested at 0, (unirradiated) 4, 24, 48 or 72 hours afterirradiation using an acid guanidinium thiocyanate-phenol-chloroformmixture, as described by Chomczynski et al. (Anal. Biochem. 162:156-9,1987). RNA harvested 24 hours after irradiation was hybridized to the 7Kmicroarray (Table 6) and to the array shown in Table 13 and analyzed asdescribed in EXAMPLE 2.

The results shown in Table 14 show the Image ID of PBL genes which weredifferentially expressed 24 hours following 2 Gy of irradiation. Theseresults indicate that cDNA microarrays, such as the 7K microarray shownin Table 6, can be used to identify genes which are differentiallyexpressed in response to irradiation. Such information can be used toidentify genes to include in a probe set (such as those shown in Table14) which will be useful for assessing potential radiation exposure in acell from an animal or plant subject. TABLE 14 Image ID Numbers ofDifferentially Expressed PBL Genes by 2 Gy 244386; 1486083; 194395;191826; 230100; 310406; 377314; 487172; 491763; 241097; 269815; 49389;511091; 626716; 154472; 753104; 205633; 810711; 77577; 130826; 769603;884425; 811015; 1475730; 826254; 80649; 82556; 825080; 213136; 898221;273546; 701112; 843312; 128126; 415899; 146605; 823665; 745019; 1391644;461759; 810017; 591653; 713193; 687054; 70692; 126466; 242790;

The alteration in gene expression following γ-irradiation was examinedin PBLs for five genes identified as being differentially expressed inresponse to irradiation (Table 14). As shown in FIG. 10, the expressionof DDB2, CIP/WAF1, XPC, cyclin G1 and PCNA genes in response toirradiation was induced 3-6 fold over non-irradiated PBLs. These resultsdemonstrate that the present invention can be used to determine if asubject has been exposed to ionizing radiation by testing PBLs, whichare sensitive to the effects of irradiation. Genes which are identifiedas being differentially expressed in response to irradiation (forexample Table 14) are incorporated into a probe set, which is exposed tothe labeled nucleic acid of a test cell (such as PBLs) using amicroarray.

A timecourse of induction following 2 Gy γ-rays delivered ex vivo toPBLs was also examined as described in EXAMPLE 3. RNA was harvested fromlymphocytes following one, two, or three days of incubation. Followingirradiation, the DDB2, CIP1/WAF1, and XP-C genes reached maximalinduction 24 hours after irradiation, then declined (FIG. 11). Theinduction of PCNA and IFNγp10 reached maximal levels 48 hours afterinduction (FIG. 11). These results demonstrate that the identificationof which genes are differentially expressed in a biological specimen,can be correlated with the approximate time of irradiation. For example,a probe set which contains the DDB2, CIP1/WAF1, and XP-C genes would beuseful for determining if a subject has been exposed within the past 24hours, and a probe set which contains the PCNA and IFNγp10 gene would beuseful for determining if a subject has been exposed within the past 48hours.

The relative induction of expression was correlated to dosage, as shownin FIGS. 12A-12D for several genes. PBL were irradiated ex vivo withgraded doses of γ-rays and RNA was harvested after four hours (FIG.12A), 24 hours (FIG. 12B), 48 hours (FIG. 12C), or 72 hours (FIG. 12D)of incubation. These results demonstrate that the measured relativeinduction of expression of genes that are differentially expressed inresponse to irradiation, can be used to interpret the results. Forexample, by determining the relative induction of the radiosensitivegenes DDB2 or WAF-1 in a particular cell type, that relative inductionis correlated to the dosage of irradiation received by a subject, suchas a plant, animal, or microbial subject.

In conclusion, this example demonstrates that biological specimens canbe used to determine if a cell has been exposed to a biologicallysignificant amount of ionizing radiation, and approximately how muchtime has elapsed since the cell was exposed. In addition, the relativeinduction of radiosensitive genes can be correlated to the dosage ofirradiation received by the cell.

EXAMPLE 10 Analysis of In Vivo Irradiated Organisms

This example describes methods which can be used to analyze organismswhich have been, or may have been, exposed to ionizing radiation.

Analysis of Irradiated Animals

The analysis of animals which have potentially been exposed to, or areknown to have been exposed to, ionizing irradiation can be performed. Astest subjects, rodents such as mice or rats are whole body irradiated,for example using a Mark I-68 ¹³⁷Cs source (JL Shepherd and Associates,San Fernando, Calif.) to deliver total doses between 2 and 500 cGy asdescribed in the above examples. Following irradiation, biologicalsamples are harvested from the animals. Examples of biological samplesinclude, but are not limited to, those listed above in EXAMPLE 9.Tissues are flash frozen in GTC (guanidine thiocyanate solution, usedabove), and the RNA is isolated and reverse transcribed incorporating alabel, as described above. The labeled nucleic acids are then exposed toan array, for example using mouse equivalents of the genes shown in anyof Tables 9-13. A test array can be generated, by selecting for probesets as described herein, for example probe sets designed to detectradiation exposure in a specimen that is to be tested, such asperipheral blood cells for example leukocytes. Patterns or hybridizationto the array can then be observed to determine the likelihood ofexposure, and/or a dose of exposure in subsequent test subjects.

Analysis of Subjects Undergoing Radiation Therapy

The disclosed arrays can also be used to evaluate and/or select patientswho are undergoing (or are candidates for) radiation therapy, for thetreatment of cancer or a tumor. For example, subjects can be monitoredduring radiotherapy to detect gene expression induced by irradiation.Differential gene expression provides diagnostic or prognosticinformation, for example, it may indicate whether sufficient doses ofradiation are being administered to cause regression of the cancer. Anindication that a sufficient dose of radiation has been used may be apattern of differential gene expression associated with a successfuloutcome of treatment of tumors of a particular type. In addition,subjects can be monitored to determine if the dose of radiation iscausing harmful effects, for example iatrogenic carcinogenesis orsuppression of the immune system by determining whether a pattern ofdifferential expression has occurred that is associated with either ofthese outcomes.

The RNA from subjects is isolated, reverse-transcribed, and labeled asdescribed above in EXAMPLE 2. The labeled nucleic acids are then exposedto an array, for example an array which contains a probe set which hasbeen selected for genes which are differentially expressed at theradiation dose which the subject is being treated with. For example, thearray may include genes which have been shown using the methodsdescribed in the above examples to be differentially expressed at 2 Gy,a typical daily dose which a subject might receive in radiotherapy. Inaddition, an array can be selected based on the tumor which is beingtreated. For example, the array can include or consist of genes whichhave been shown (using the methods described in the above examples) tobe differentially expressed at the dose which the subject is receiving,in the type of tumor the subject suffers from. Such specific probe setsprovide useful diagnostic information regarding the subject's progressin the radiotherapy.

Analysis of Irradiated Plants or Microbes

In addition to humans and other animals, plants or microbes which havebeen, or may have been, exposed to radiation can be analyzed using thepresent invention. In one example, following a nuclear accident, ifthere are no animals present to examine to determine the amount ofexposure, the plant or microbial flora present in the area can beanalyzed. Nucleic acids, for example RNA is isolated using standardmethods. Nucleic acids are isolated from any part of the plant, forexample roots, stems, leaves or flowers. The RNA is isolated,reverse-transcribed, and labeled as described above. The labeled plantnucleic acids can then be exposed to an array, for example using plantequivalents of the genes shown in Tables 9-13, or other plant genes thatare found to be differentially expressed following radiation exposure.

Having illustrated and described the principles of assessing whether anorganism has been exposed to biologically significant levels of ionizingradiation by detecting differential cellular expression of genes thatare differentially expressed following such exposure, it should beapparent to one skilled in the art that the invention can be modified inarrangement and detail without departing from such principles. In viewof the many possible embodiments to which the principles of ourinvention may be applied, it should be recognized that the illustratedembodiments are only examples of the invention and should not be takenas a limitation on the scope of the invention. Rather, the scope of theinvention is in accord with the following claims. We therefore claim asour invention all that comes within the scope and spirit of theseclaims.

1. A method of identifying cells that have been exposed to radiation induced biological stress, comprising: exposing a probe set to a labeled nucleic acid composition from a test cell which specifically hybridizes to members of the probe set when the cell has been exposed to an amount of ionizing radiation sufficient to cause differential expression of stress genes in the cell; and determining whether the nucleic acid composition hybridizes to the nucleic acid molecules representing genes that are differentially expressed.
 2. The method of claim 1, wherein the probe set is bound in an array to a surface.
 3. The method of claim 2, wherein the probe set comprises oligonucleotides that specifically hybridize to sequences in the nucleic acid composition.
 4. The method of claim 3, wherein the nucleic acid composition comprises cDNA reverse transcribed from mRNA in the test cell.
 5. The method of claim 1, wherein the labeled nucleic acid is labeled with a fluorophore.
 6. The method of claim 1, further comprising exposing the probe set to a labeled nucleic acid composition from a control cell, such that the labeled nucleic acid composition from the control cell specifically hybridizes to members of the probe set.
 7. The method of claim 1, further comprising determining a probable time of radiation exposure, wherein the probe set comprises probes that specifically hybridize to the labeled nucleic acid composition, when the nucleic acid composition has been obtained more than four hours after exposure to the biologically significant amount of ionizing radiation, and wherein hybridization to the probe set indicates that the cells have been exposed to the biologically significant amount of ionizing radiation at least four hours previously.
 8. The method of claim 1, further comprising determining a probable time of radiation exposure, wherein the probe set comprises probes that specifically hybridize to the labeled nucleic acid composition, when the nucleic acid composition has been obtained more than 24 hours after exposure to the biologically significant amount of ionizing radiation, and wherein hybridization to the probe set indicates that the cells have been exposed to the biologically significant amount of ionizing radiation at least 24 hours previously.
 9. The method of claim 1, further comprising determining a probable time of radiation exposure, wherein the probe set comprises probes that specifically hybridize to the labeled nucleic acid composition, when the nucleic acid composition has been obtained more than 48 hours after exposure to the biologically significant amount of ionizing radiation and wherein hybridization to the probe set indicates that the cells have been exposed to the biologically significant amount of ionizing radiation at least 48 hours previously.
 10. The method of claim 1, wherein the probe set comprises probes that specifically hybridize to the labeled nucleic acid composition, when the nucleic acid composition has been exposed to less than about 25 cGy of ionizing radiation.
 11. The method of claim 1, wherein the probe set comprises genes that are differentially expressed by at least 1.5 fold following exposure to a biologically significant amount of ionizing radiation.
 12. The method of claim 11, wherein the genes are differentially expressed by at least 2-fold following exposure to a biologically significant amount of ionizing radiation.
 13. The method of claim 1, wherein the probe set comprises: (a) at least: 10% of the probes identified in any one of Tables 9, 10, 11, 12, 13 or 14; or (b) at least 10 of the probes identified in any one of Tables 9, 10, 11, 12, 13 or 14; or (b) at least 20 of the probes identified in any one of Tables 9, 10, 11, 12, 13 or
 14. 14.-20. (canceled)
 21. A probe set, comprising at least 10% of the probes identified in any one of Tables 9, 10, 11, 12, 13 or
 14. 22.-23. (canceled)
 24. A method of identifying cells that have been exposed to radiation induced biological stress, comprising: providing a plurality of probe elements bound to a surface, each probe element comprising a nucleic acid that is differentially expressed by a cell following ionizing radiation induced biological stress; contacting the plurality of probe elements with a plurality of test nucleic acid sequences obtained from a test cell, under conditions that allow the test nucleic acid sequences to specifically hybridize to one or more of the probe elements, and provide a signal which indicates differential expression of one or more genes if the cell has been exposed to an amount of ionizing radiation sufficient to cause differential expression of stress genes in the cell; and detecting the presence or absence of the signal.
 25. The method of claim 24, wherein the plurality of probe elements is a selected set of nucleic acids from a set of nucleic acids that hybridize to nucleic acids obtained from cells exposed to ionizing radiation.
 26. The method of claim 24, wherein the probe elements are nucleic acid sequences that are differentially expressed by a cell more than four hours after exposure to the ionizing radiation.
 27. The method of claim 26, wherein the probe elements are nucleic acid sequences that are differentially expressed at a time at least 24 hours after exposure to the ionizing radiation.
 28. The method of claim 26, wherein the probe elements are nucleic acid sequences that are differentially expressed more than 48 hours after exposure to the ionizing radiation.
 29. The method of claim 26, wherein the probe elements comprise nucleic acid sequences that are differentially expressed by at least 1.5-fold following exposure to a biologically significant amount of ionizing radiation.
 30. The method of claim 29, wherein the nucleic acid sequences are differentially expressed by at least 2-fold following exposure to a biologically significant amount of ionizing radiation. 31.-32. (canceled)
 33. The method of claim 1, wherein the plurality of probe elements comprises nucleic acid elements identified by Image ID numbers: 26474, 268652, 34396, 35318, 50615, 110589, 203132, 247483, 251591, 360778, 415817, 469954, 489805, 753447, 884719, or
 1493160. 34. The method of claim 1, wherein the probe nucleic acids are DNA.
 35. The method of claim 34, wherein the probe nucleic acids are cDNA.
 36. The method of claim 35, wherein the test nucleic acids are cDNA obtained from mRNA expressed by the test cell.
 37. The method of claim 1, wherein the probe nucleic acids are about 10 to about 1,000,000 nucleotides in length.
 38. The method of claim 1, further comprising contacting the plurality of probe elements with a plurality of control nucleic acids obtained from mRNA of a control cell that has not been exposed to a biologically significant amount of ionizing radiation; and determining whether the nucleic acids from mRNA of the control cells hybridize differentially to the probe elements than the nucleic acid composition from the test cell.
 39. The method of claim 24, wherein the test nucleic acid sequences are labeled with a fluorophore that detects hybridization of the test nucleic acid sequences to the target sequences.
 40. The method of claim 39, wherein the test nucleic acid sequences are labeled with a first label that detects hybridization of the test nucleic acids to the probe sequences, and the control nucleic acids are labeled with a second label that detects hybridization of the control nucleic acids to the probe sequences, and the first and second labels interact to indicate whether expression of a nucleic acid in the test cell has increased or decreased, relative to expression of the control nucleic acids.
 41. The method of claim 40, wherein the first and second labels are fluorophores of different colors.
 42. (canceled)
 43. The method of claim 24, wherein the cells are animal cells, microbial cells, or plant cells.
 44. The method of claim 43 wherein the animal cells are human cells.
 45. The method of claim 43, wherein the animal cells are peripheral blood cells.
 46. The method of claim 45, wherein the peripheral blood cells are peripheral blood mononuclear cells.
 47. The method of claim 46, wherein the peripheral blood mononuclear cells are lymphocytes.
 48. (canceled)
 49. A method of making a microarray for identifying cells that have actually or potentially been exposed to an amount of ionizing radiation sufficient to cause differential expression of stress genes in the cell, comprising: identifying genes that are differentially expressed by a cell following exposure to the ionizing radiation; and providing a plurality of probe elements bound to a substrate surface, the probe elements comprising probe nucleic acids from genes that are identified as differentially expressed by a cell following ionizing radiation induced biological stress, and wherein the probe nucleic acid is capable of hybridizing to a nucleic acid differentially expressed by the cell following exposure of the cell to the ionizing radiation.
 50. The method of claim 49, wherein identifying genes that are differentially expressed by a cell following exposure to biologically significant amounts of ionizing radiation comprises exposing the cell to a biologically significant amount of ionizing radiation, obtaining mRNA expressed by the cell, reverse transcribing the mRNA into cDNA, labeling the cDNA, and hybridizing the labeled cDNA to a probe set and identifying members of the probe set that hybridize with the labeled cDNA.
 51. The method of claim 50, where in the genes comprise p53 regulated genes.
 52. The method of claim 49, further comprising determining a dose response relationship between radiation exposure and differential expression of one or more genes.
 53. A method of diagnosing ionizing radiation exposure in a subject, comprising: obtaining a biological sample from the subject; synthesizing cDNA from mRNA expressed in one or more cells of the biological sample, and labeling the cDNA with a detectable label; exposing the labeled cDNA to a probe set which represents genes that are differentially expressed in the biological sample following exposure to ionizing radiation, and determining if the labeled cDNA selectively hybridizes to one or more probes of the probe set that are associated with the radiation exposure.
 54. The method of claim 53, wherein the probe set comprises one or more of the probes identified in any one of Tables 9, 10, 11, 12, 13 or
 14. 55.-57. (canceled)
 58. The method of claim 49, wherein identifying genes that are differentially expressed comprises identifying genes that are differentially expressed in a cell type that is to be obtained from a subject for testing. 59.-61. (canceled)
 62. The method of claim 53, further comprising detecting patterns of differential expression associated with biologically significant radiation exposure.
 63. (canceled)
 64. A method for measuring a biological response to in a subject comprising: obtaining a biological sample from the subject; synthesizing cDNA from mRNA expressed in one or more cells of the biological sample, labeling the cDNA with a detectable label; and exposing the labeled cDNA to a probe set; and determining if the labeled cDNA selectively hybridizes to one or more probes of the probe set differentially expressed following exposure to ionizing radiation.
 65. The method of claim 64 wherein the subject is undergoing radiotherapy for the treatment of cancer and the probe set is used to monitor the subject's biological response to the radiotherapy.
 66. (canceled)
 67. The method of claim 64 wherein the biological response is a biological response to potential radiation exposure in the subject. 68.-69. (canceled)
 70. The method of claim 67, wherein the biological response is measured in a cell type.
 71. The method of claim 70, wherein the cell type is a peripheral blood cell obtained from the subject.
 72. The method of claim 64, further comprising determining a relationship between radiation exposure and differential expression of one or more genes to determine a probable radiation dose in cells that have actually or potentially been exposed to the ionizing radiation.
 73. The method of claim 1, wherein the probe set comprises SEQ ID NO: 7, 9, 10 or
 28. 74. The method of claim 1, wherein at least 50% of the nucleic acid sequences of the probe set are differentially expressed following exposure to the ionizing radiation. 